UMD Theses and Dissertations
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Item SEROTONIN REGULATES AN OLFACTORY CRITICAL PERIOD IN DROSOPHILA(2024) Mallick, Ahana; Araneda, Ricardo; Gaudry, Quentin; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Serotonin (5-HT) is known to modulate early development during critical periods when experience drives heightened levels of plasticity in sensory systems. Studies in the somatosensory and visual cortices implicate multiple target points of serotonergic modulation, yet the underlying cellular and molecular mechanisms of 5-HT modulation of critical period plasticity remain elusive. Here, we take advantage of the genetically tractable olfactory system of Drosophila to investigate how 5-HT modulates critical period plasticity (CPP) in the CO2 sensing circuit of fruit flies. During the critical period, chronic exposure to CO2 has been shown to increase the volume of the CO2 sensing V glomerulus. We found that 5-HT release by serotonergic neurons in the antennal lobe (AL) is required for increase in the volume of the V glomerulus. Furthermore, signaling via the 5-HT1B, 5-HT2B and 5-HT7 receptors in different neuronal populations is also required during the critical period. Olfactory CPP is known to involve local inhibitory networks and consistent with this we found that knocking down 5-HT7 receptors in a subset of GABAergic local interneurons was sufficient to block CPP, as was knocking down GABA receptors expressed by olfactory sensory neurons (OSNs). Additionally, 5-HT2B expression in the cognate OSNs sensing CO2 is also essential for CPP indicating that direct modulation of OSNs also contributes to the olfactory CPP. Furthermore, 5-HT1B expression by serotonergic neurons in the olfactory system is also required during the critical period. Our study reveals that 5HT modulation of multiple neuronal targets is necessary for experience-dependent structural changes in an odor processing circuit. Finally, we wanted to isolate the neuromodulatory effects of individual serotonergic neurons. To achieve this, we combined a state-of-the-art technique to sparsely label serotonergic neurons and a computer algorithm to search against 10,000 Gal4 promoter lines and identify candidate lines that would allow individual manipulation of the 110 serotonergic neurons.Item Quantifying the relative contribution and furthering qualitative understanding of ftz cis-regulatory elements in Drosophila melanogaster(2022) Fischer, Matthew Douglas; Pick, Leslie; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Embryonic development is coordinated by interactions within gene regulatory networks. This process is orchestrated at the level of transcription through the regulatory properties of enhancers, which direct spatiotemporal expression patterns when bound by specific trans-acting factors. Though enhancers can act upon promoters located at great distances irrespective of orientation, the contributions from these cis-regulatory elements (CREs) are limited by insulators and/or tethering elements that organize chromatin architecture. Much research has been conducted towards understanding the coordination of the segmentation genes that pattern the basic body plan of the fruit fly, Drosophila melanogaster, during embryogenesis. The pair-rule genes (PRGs) of this pathway, such as fushi tarazu (ftz), are expressed in seven alternating stripes across the embryo. These PRGs are required for the development of body segments, and the mis-regulation of a single transcriptional domain can result in the loss of a segment. Here, I have investigated the ftz CREs to more precisely determine their sufficiency to direct expression within ftz stripe domains and their necessity for doing so in the native context of the gene. To investigate the sufficiency, I have generated 36 standardized reporter transgenes from 18 CREs, tested in both forward and reverse orientations. All CREs examined have been inserted into the same XbaI site of the reporter plasmid, and the transgenes have been inserted into the same genomic region. Through in situ hybridization experiments, I have determined that the qualitative patterns conferred by every CRE is orientation-dependent, and I have identified two putative insulators and/or tethering elements, proposed to explain this observation. To investigate their necessity, I targeted four genomic regulatory regions for precise deletion using the CRISPR/Cas9 system to generate seven deletion mutants. Though deletions were expected to cause lethality, most of the mutants are homozygous viable and fertile; only a mutant simultaneously removing two seven-stripe CREs was homozygous lethal. Quantitative gene expression analysis by fluorescent in situ hybridization chain reaction revealed that there is a critical threshold of ftz abundance required in each stripe for segmentation to proceed. In conclusion, I have determined that the ftz CREs are redundant and function together in a non-additive manner.Item PHYSIOLOGICAL CHARACTERIZATION OF SPECIFIC LOCAL INTERNEURON SUBPOPULATIONS IN THE DROSOPHILA ANTENNAL LOBE(2022) Schenk, Jonathan Edward; Gaudry, Quentin; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The olfactory system of the fruit fly Drosophila melanogaster is an invaluable model for understanding circuit function. Composed mainly of olfactory receptor neurons (ORNs), projection neurons (PNs), and local interneurons (LNs), it is an analogous structure to mammalian olfactory systems. Of these cell types, LNs are particularly intriguing. These neurons are found in a variety of morphologies and with differing neurotransmitter and receptor profiles. Given their heterogeneity, it is critical to gain an understanding of their roles in olfactory circuits. In this work, I probe the physiology and functions of two unique subpopulations of LNs in the antennal lobe (AL). In the first population, I demonstrate LNs which respond to extrasynaptic, paracrine levels of serotonergic modulation. These LNs then engage in postsynaptic inhibition and subtractive gain control, which is contrary to typical LNs. The second population I characterize are previously undescribed nonspiking LNs in the fly AL. Nonspiking cells are common to insect olfaction as well as other sensory pathways in vertebrates. I find that these neurons are likely to be electrotonically compartmentalized, such that activation within individual regions does not propagate across the whole cell, suggesting roles in previously unexplained mechanisms such as intraglomerular inhibition. The results of this work suggest more heterogeneity in Drosophila LNs than previously assumed and cements the importance of interneuron contribution to neuronal function.Item SEQUENTIAL EXPRESSION OF NICOTINIC ACETYLCHOLINE RECEPTOR SUBUNITS SUPPORTS DEVELOPMENT AND PLASTICITY OF A DROSOPHILA CENTRAL SYNAPSE(2021) Rosenthal, Justin Samuel; Pick, Leslie LP; Yuan, Quan QY; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The central nervous system (CNS) of animals is arguably one of the most sophisticated instruments designed by nature, and one its cardinal components is the postsynaptic specialization. Decades of studies on the cholinergic neuromuscular junction and the central glutamatergic synapses in vertebrate organisms has informed us of just how many factors are at play during postsynaptic development. However, despite its importance, the central cholinergic synapse is one system lacking the same knowledge base as the above models. The thesis work presented here was designed with the aim of understanding if and how nicotinic acetylcholine receptor (nAchR) activity at the postsynapse is used by a developing neuron to shape the structural and functional properties of the synapse and its dendrite arbor during normal periods of maturation. To this end, we employed the ventral lateral neuron (LNv) as a cellular model for Drosophila CNS development. This small group of cells are second-order projection neurons which convey visual activity to higher brain centers and are also critical mediators of adjusting the fly’s internal circadian clock. We report how nAchRs not only play a role in LNv neurophysiology by the end of larval development but show how in fact they actively participate during the formation and refinement of the LNv postsynapse. Our transcriptomic, morphological and physiological approaches reveal that two functionally distinct nAchR subunit genes, Da1 and Da6, are preferentially expressed during separate periods of larval development. Here, young and immature LNvs are characterized by high Da6 expression which facilitates synaptic formation. As the animal grows, Da6 is downregulated and Da1 is upregulated, which is necessary for synaptic stabilization and maturation. We also expand the scope of our primary investigation by identifying promising candidate genes, including transcription factors, molecular chaperones and membrane-associated proteins, that are key to orchestrating the successive stages of nAchR expression, maturation and postsynaptic activity. In summary, our findings will work to clarify in vivo subunit-specific functionalities for the insect nAchR and illustrate how individual nAchR subunits in the CNS are coordinately regulated within a single cell through time to actively regulate distinct properties of the synapse during development.Item Comparative transcriptomics of long intergenic noncoding RNAs in Drosophila(2015) Nyberg, Kevin Glenn; Machado, Carlos A; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Without the constraints of the amino acid code, long intergenic noncoding RNAs (lincRNAs) can be expected to evolve along different trajectories than protein-coding genes. Most studies of lincRNA evolution analyze evolution only at the sequence level without ascertaining whether the lincRNA is expressed. Over 2,000 lincRNAs (and counting) have already been identified in the classic model system Drosophila melanogaster. Here, using RNA-Seq and computational identification of protein-coding ability, we identify 1,768 lincRNA transcripts at 1,586 unique loci in a second species of Drosophila - D. pseudoobscura. These lincRNAs are expressed in every surveyed developmental stage (1st instar larva, 3rd instar larva, pupa, and adult) in both sexes, with a large number increasing in expression as male development proceeds. This male bias can largely be explained by overrepresentation of lincRNAs in the testes. Unequal distributions of sex-biased lincRNAs on the X chromosome and autosomes are consistent with selection-based models of gene trafficking on or off the X chromosome, implying function for some of these lincRNAs. Finally, reciprocal blast searches between annotated lincRNAs in the D. pseudoobscura and D. melanogaster transcriptomes identify 80 conserved lincRNAs. Interestingly, direct coordinate conversions between the two genomes reveal another 54 D. pseudoobscura lincRNAs that are expressed in the same position as a D. melanogaster lincRNA but have low enough sequence conservation to preclude alignment via blast. Whether these positionally equivalent lincRNAs are true homologs with similar functions in both genomes is unclear, but we look at other transcript features, such as transcript orientation, gene structure, and developmental expression profiles to explore this possibility. We find 22 high-confidence lincRNA homologs with conservation of multiple transcript-level features, and we designate these as high-confidence homologs that warrant further biological investigation. This work represents the first comparative transcriptomic analyses of lincRNAs in Drosophila.Item Phagocytosis and signaling in the innate immune system(2012) Gonzalez, Elizabeth Anne Cates; Wu, Louisa P; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The innate immune response provides broad spectrum defense through germline encoded components. Many aspects of innate immunity, such as the activation of NFκB transcription factors and phagocytosis, are highly conserved within the animal kingdom. The innate immune response of the cow, in particular, is important due to the cow's agricultural value. A major proportion of acute disease in domestic cattle is caused by Gram-negative bacteria, which produce the outer membrane component lipopolysaccharide (LPS). LPS binds to Toll-like receptor (TLR) 4 and activates multiple signaling pathways, which have been well-studied in humans, but not in ruminants. Human myeloid differentiation-factor 88 (MyD88) and TIR-domain containing adaptor protein (TIRAP) are critical proteins in the LPS-induced NFκB and apoptotic signaling pathways in humans. We demonstrated through the expression of dominant negative constructs in bovine endothelial cells that both MyD88 and TIRAP activate NFκB in the cow. Additionally, bovine TIRAP was also shown to transduce LPS–induced apoptosis, indicating that multiple aspects of the TLR4–dependent signaling pathways are conserved between cows and humans. The model organism Drosophila melanogaster, was subsequently utilized to investigate the role of another branch of the innate immune response: phagocytosis. The extracellular fluid surrounding phagocytic cells in Drosophila has a high concentration of the amino acid glutamate. While glutamate has been well-characterized as a neurotransmitter, its effect, if any, on immune cells is largely unknown. We identified that a putative glutamate transporter in D. melanogaster, polyphemus (polyph), is critical to the fly's immune response. Flies with a disrupted polyph gene exhibit decreased phagocytosis of microbial-derived bioparticles but not of latex beads. Additionally, polyph flies show increased susceptibility to S.aureus infection, decreased induction of the antimicrobial peptide (AMP) Cecropin, increased melanization response, and increased ROS production. Glutamate transport has previously been shown to regulate the synthesis of the antioxidant glutathione. We demonstrate that a polyph–dependent redox system is necessary to maintain the immune cells' function against an infection. By utilizing two species, the cow and the fly, to study the innate immune system, we have gained unique and novel insights into NFκB activation and phagocytosis.Item RNA SILENCING AND HIGHER ORDER CHROMATIN ORGANIZATION IN DROSOPHILA(2011) Moshkovich, Nellie; O'Brochta, David A; Lei, Elissa P; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Higher order chromatin organization influences gene expression, but mechanisms by which this phenomenon occurs are not well understood. RNA silencing, a conserved mechanism that involves small RNAs bound to an Argonaute protein, mediates gene expression via transcriptional or post-transcriptional regulation. Recently, a role for RNA silencing in chromatin has been emerging. In fission yeast, a major role of RNA interference (RNAi) is to establish pericentromeric heterochromatin. However, whether this mechanism is conserved throughout evolution is unclear. In Drosophila, a powerful model organism, there are multiple functionally distinct RNA silencing pathways. Previous studies have suggested the involvement of the Piwi-interacting RNA (piRNA) and endogenous small interfering RNA (endo-siRNA) pathways in heterochromatin formation in order to silence transposable elements in germline and somatic tissues, respectively, but direct evidence is lacking. We addressed whether the genomic locations generating these small RNAs may act as AGO-dependent platforms for heterochromatin recruitment. Our genetic and biochemical analyses revealed that heterochromatin is nucleated independently of endo-siRNA and piRNA pathways suggesting that RNAi-dependent heterochromatin assembly may not be conserved in metazoans. Chromatin insulators are regulatory elements characterized by enhancer blocking and barrier activity. Insulators form large nuclear foci termed insulator bodies that are tethered to the nuclear matrix and have been proposed to organize the genome into distinct transcriptional domains by looping out intervening DNA. In Drosophila, RNA silencing has been reported to affect nuclear organization of gypsy insulator complexes and formation of Polycomb repression bodies. Our studies revealed that AGO2 is required for CTCF/CP190-dependent Fab-8 insulator function independent of its catalytic activity or Dicer-2. Moreover, AGO2 associates with euchromatin but not heterochromatin genome-wide. Also, AGO2 associates physically with CP190 and CTCF, and mutation of CTCF, CP190, or AGO2 decreases chromosomal looping interactions and alters gene expression. We propose a novel RNAi-independent role for AGO2 in the nucleus. We postulate that insulator proteins recruit AGO2 to chromatin to promote or stabilize chromosomal interactions crucial for proper gene expression. Overall, our findings demonstrate novel mechanisms by which RNA silencing affects gene expression on the level of higher order chromatin organization.Item JUVENILE HORMONE BIOSYNTHESIS AND SIGNALING PATHWAY IN DROSOPHILA MELANOGASTER(2011) Mahmoud Abdou, Mohamed Ali; Wang, Jian; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Insect development and metamorphosis are controlled by two major hormones; 20-hydroxyecdysone (20E) and juvenile hormone (JH). 20E signaling pathway is well recognized while JH signaling is still ambiguous. For a better understanding of JH biosynthesis and signaling we worked on two parallel projects; reverse genetic and forward genetic studies. In the reverse genetic study, we have tested the potential functional redundancy between Methoprene-tolerant (Met) and germ cell-expressed (gce), two paralog bHLH-PAS transcription factors in Drosophila that were suggested to be JH receptors. Met null mutants are viable, resistant to JH and low fecundity. No gce mutant was available at the begening of this project. We generated a gce null allele and found that it phenocopies Met mutants. Met-gce double mutants are lethal at prepupal stage, which is similar to the JH-deficient flies. Krüppel homolog1 (Kr-h1) and broad (br) are two known JH signaling componets. Further investigations revealed that Met-gce double mutant diminishes Kr-h1 expression, induces precocious br expression, and causes premature and enhanced caspase-dependent programmed cell death. Therefore, we conclude that Met and Gce are functionally redundant in transducing JH signals. Expression of br is induced by 20E, but its induction can be suppressed by JH. In the forward genetic study, we designed and conducted a novel genetic screen to isolate mutations that can de-repress br expression at early larval stages. From 4,400 lethal lines, 55 mutations were isolated based on the precocious br expression in 2nd instar larvae. Genes associated with these 55 mutations include apterous, InR, NMAR1, Fpps and Kr-h1, which are known to be involved in JH biosynthesis or signaling. Other genes encode proteins with various molecular functions, including enzymes, signal transduction molecules, and transcriptional factors. Among them, there are three Wnt signaling components, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd) and two TGF-β signaling components, thick vein (tkv) and mothers against Dpp (mad). We further demonstrated that Wnt signaling mediates JH signaling by regulating Met and gce expression, and that TGF-β signaling controls JH biosynthesis by upregulating transcription of JH acid methyltransferase (jhamt), a key regulatory enzyme of JH biosynthesis.Item Characterization of Atg6 function in autophagy and growth control during Drosophila melanogaster development(2010) Hill, Jahda Hope; Wu, Louisa P; Baehrecke, Eric H; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The tumor suppressor Beclin 1 mitigates cell stress by regulating the lysosomal degradation pathway known as autophagy. This process involves formation of intracellular double-membraned vesicles, known as autophagosomes, which engulf proteins and damaged organelles and fuse with lysosomes, where the contents are degraded. It is unclear whether the function of Beclin 1 in autophagy is related to cell transformation in beclin 1+/- animals. Using the fruit fly, Drosophila melanogaster, I investigated the function of the Beclin 1 ortholog Atg6 in autophagy and growth control. Through transgenic experiments, I found that Atg6, like Beclin 1, induces autophagy by functioning in a complex consisting of the lipid kinase Vps34 and the serine–threonine kinase Vps15. I also generated a strong loss of function mutant, Atg61, and found that Atg6 is required for development. Atg6 mutant animals contained an excess of blood cells, which surrounded melanotic tumors prior to death. At the cellular level, Atg6 is required for autophagy and endocytosis, and cells lacking Atg6 accumulate high levels of the endoplasmic reticulum stress protein Hsc3 and the adaptor protein p62. I also showed that Atg6 mutant cells displayed mis-regulated nuclear localization of NF κB proteins, transcription factors whose downstream targets include regulators of innate immunity. Significantly, my results suggest that Atg6 may regulate growth independent of its function in autophagy, as mosaic loss of Atg6 in the eye resulted in over-representation of Atg6 mutant cells, a phenotype not shared by other autophagy gene mutant mosaics. Finally, through a collaborative effort, our lab identified a novel function for Atg6 in regulation of epithelial cell polarity. This finding is significant, as epithelial tumor cells are known to lose polarity during metastasis. Our studies have provided a significant contribution to the Beclin 1 field, by providing the first characterization of a Drosophila Atg6 mutant, and demonstrating its function in novel cellular processes.Item Role and Regulation of Autophagy During Developmental Cell Death in Drosophila Melanogaster(2010) McPhee, Christina Kary; Mount, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Two prominent morphological forms of programmed cell death occur during development, apoptosis and autophagic cell death. Improper regulation of cell death can lead to a variety of diseases, including cancer. Autophagy is required for survival in response to starvation, but has also been associated with cell death. It is unclear how autophagy is regulated under specific cell contexts in multi-cellular organisms, and what may distinguish autophagy function during cell survival versus cell death. Autophagic cell death is characterized by cells that die in synchrony, with autophagic vacuoles in the cytoplasm, and phagocytosis of the dying cells is not observed. However, little is known about this form of cell death. Autophagic cell death is observed during mammalian development, during regression of the corpus luteum and involution of the mammary and prostate glands. Autophagic cell death is also observed during development of the fruitfly Drosophila melanogaster, during larval salivary gland cell death. Drosophila is an excellent genetic model system to study developmental cell death in vivo. Cells use two main catabolic processes to degrade and recycle cellular contents, the ubiquitin/proteasome system (UPS) and autophagy. Here I investigate the role of the UPS and autophagy in developmental cell death using Drosophila larval salivary glands as an in vivo model. Proteasome inhibitors are being used in anti-cancer therapies; however the cellular effects of proteasome inhibition have not been studied in vivo. Here I demonstrate that the UPS is impaired during developmental cell death in vivo. Taking a proteomics approach to identify proteins enriched in salivary glands during developmental cell death and in response to proteasome impairment, I identify several novel genes required for salivary gland cell death, including Cop9 signalsome subunit 6 and the engulfment receptor Draper. Here I show that the engulfment receptor Draper is required for salivary gland degradation. This is the first example of an engulfment factor that is autonomously required for self-clearance. Surprisingly, I find that Draper is cell-autonomously required for autophagy during cell death, but not for starvation-induced autophagy. Draper is the first factor to be identified that genetically distinguishes autophagy that is associated with cell death from cell survival.