Browsing by Subject "Drosophila melanogaster"
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Item A Comparative Study of Lethal-Bearing Heterozygotes in a Local Population of Drosophila Melanogaster(Texas Tech University, 1972-08) Robinson, Robert VaughnNot Available.Item A FGF-Hh feedback loop controls stem cell proliferation in the developing larval brain of drosophila melanogaster(2009-05-15) Barrett, Andrea LynnThe adult Drosophila central nervous system is produced by two phases of neurogenesis: the first phase occurs during embryonic development where the larval brain is formed and the second occurs during larval development to form the adult brain. Neurogenesis in both phases is caused by the activation of neural stem cell division and subsequent progenitor cell division and terminal differentiation. Proper activation of neural stem cell division in the larval brain is essential for proper patterning and functionality of the adult central nervous system. Initiation of neural stem cell proliferation requires signaling from the Fibroblast Growth Factor (FGF) homolog Branchless (Bnl) and by the Hedgehog (Hh) growth factor. I have focused on the interactions between both of these signaling pathways with respect to post-embryonic neural stem cell proliferation using the Drosophila larval brain. Using proliferation assays and quantitative real-time PCR, I have shown that Bnl and Hh signaling is inter-dependent in the 1st instar larval brain and activates neural stem cell proliferation. I have also shown that overexpression of bnl can rescue signaling and neuroblast proliferation in a hh mutant. However, overexpression of hh does not rescue signaling or neuroblast proliferation in a bnl mutant, suggesting that Bnl is the signaling output of the Bnl-Hh feedback loop and that all central brain and optic lobe neural stem cells require Bnl signaling to initiated proliferation.Item Alternative Processing of SREBP in Site 2 protease and Scap mutants during larval development in Drosophila(2009-01-14) Matthews, Krista Ann; Rawson, Robert B.Lipid metabolism is regulated by the membrane-bound transcription factor, sterol regulatory element binding protein (SREBP). SREBP requires release of the amino terminus from the membrane to activate transcription of genes involved in cholesterol and fatty acid synthesis. In response to low sterol levels, Scap escorts SREBP from the ER to the Golgi where it is cleaved by Site-1 and Site-2 proteases. The SREBP pathway is conserved in Drosophila despite these organisms being cholesterol auxotrophs. dSREBP is essential for activating genes involved in the uptake and synthesis of fatty acids which are required for rapid growth during larval development. I have demonstrated that processing of SREBP in Drosophila does not require the S2P or Scap, in contrast to the mammalian system. Flies lacking dS2P are viable and still process dSREBP. dS2P homozygotes were subviable, only emerging at 40% of the expected ratio. This phenotype can be rescued completely by supplementation with fatty acids. dSREBP activity was detected in the fat body of dS2P mutant larvae and to a lesser extent in the oenoctyes. Additionally, SREBP target genes were expressed at higher levels in dS2P homozygotes compared to dSREBP mutants, though less than wild type. dS2P mutants were viable due to alternative cleavage of dSREBP within the juxtamembrane region by the effector caspase, Drice. Flies lacking both dS2P and Drice, or the initiator caspase Dronc, exhibited an early larval lethality that could be rescued by lipid supplementation. Caspase cleavage was dependant upon the aspartic acid at residue 386 in dSREBP. dScap was not essential for larval growth or dSREBP processing in Drosophila. dScap mutants were relatively healthy, emerging at 70% of the expected numbers. dSREBP was actively cleaved in midgut and oenocytes, but significantly reduced in fat body. Levels of dSREBP mRNA and precursor were reduced in larvae lacking dScap, thus demonstrating that Drosophila SREBP is subject to feed-forward activation of its own transcription. Addition of soy lipids suppress dSREBP processing in dScap mutants, but whether this regulation is translational or post-translational is unknown. Furthermore, flies lacking both dScap and dS2P are viable, but survive less well than either single mutant alone. Membrane-bound intermediate dSREBP accumulates in double mutants, suggesting that dSREBP is processed normally by dS1P and dS2P in dScap single mutants. Thus, dScap mutants escape the larval lethality seen in dSREBP mutants due to alternative processing of dSREBP, but through different mechanism than that seen in dS2P mutants.Item Characterization of Ataxin 2-Binding Protein 1 during Female Germ Cell Differentiation in Drosophila Melanogaster(2010-11-02T18:20:48Z) Yilmaz, Omur; Buszczak, MichaelIn the Drosophila ovary, the molecules that promote the continued stepwise development of germline stem cells after their exit the niche remain largely unknown. During Drosophila oogenesis, a germline stem cell (GSC) divides asymmetrically to produce a renewing stem cell and a differentiated daughter that will progress through different stages of oogenesis to produce a mature egg. Numerous factors regulate the balance between GSC and a daughter cell, the cystoblast (CB). The Dpp/BMP signaling pathway from the niche silences bam transcription, a key differentiation factor, in the GSC. Bam protein is expressed in the cystoblast and forms a complex with its partner Bgcn, to antagonize the Pumilio-Nanos (Pum-Nos) complex through nos 3’UTR. Repression of Pum-Nos activity by the Bam-Bgcn complex permits CB differentiation. The CB goes through four incomplete divisions to give rise to a 16 cell cyst that buds off from the germarium to form an egg chamber. A number of genes have been found to have roles at 16 cell stage germ cell cysts but the molecular events that govern the intermediate stages (2-, 4- and 8-cell cysts) have remained elusive. In this study, I investigated the function of Drosophila homolog of the human disease gene Ataxin 2-binding protein 1 (A2BP1) during germline cyst differentiation. Through phenotypic analysis I showed that strong A2BP1 mutants display cystic tumors and mitosis to meiosis transition defects while weaker alleles have germline counting defects. Also, by genetic and biochemical analysis, I found that A2BP1 interacts with itself and Bruno, a known translational repressor. In addition, I examined the relationship between A2BP1 protein and other differentiation genes by expression analysis and showed that A2BP1 expression bridges the expression of the early differentiation factor Bam with late markers such as Bruno, Orb and Rbp9. The expression of A2BP1 is lost in bam, snf and mei-P26 mutants but is still present in rbp9 and arrest mutants. These observations might indicate existence of a linear hierarchy between these differentiation genes. In summary, my studies revealed A2BP1 defines a new tier within the genetic hierarchy that promotes the differentiation of single germ cells into mature 16-cell cysts during Drosophila oogenesis.Item Characterization of Drosophila SCAP: Analysis of Mutants and Evidence for a Retention Factor(2011-08-26T17:34:23Z) Ozdemir, Cafer; Rawson, Robert B.The SREBP pathway is one of the major regulators of lipid homeostasis and it is highly conserved among metazoans. SREBP is a transcription factor whose precursor is an endoplasmic reticulum (ER) transmembrane protein. In order to be activated it must travel to the Golgi apparatus via interaction with an escort protein, Scap. Scap, in turn can interact with components of the coatamer protein complex II (COPII) when lipid levels fall. In the Golgi, SREBP is cleaved sequentially by two proteases, S1P and S2P. By contrast to mammalian cells, which cannot survive without S2P or Scap, flies lacking Scap or S2P can activate SREBP. These mutants survive owing to non-canonical mechanisms of SREBP activation. Scap has a intrinsic tendency to travel to Golgi. In vertebrates, the ER retention factor, Insig, anchors the Scap:SREBP complex to the ER membrane when de novo lipid synthesis is not required. In Drosophila dSREBP pathway there is no Insig orthologues. However, our data suggest that there should be an analogous component that retains dScap in the ER. In order to discover the putative retention factor and other modifiers of the dSREBP, I set up a high through-put genome-wide screen. Employing luciferase as reporter, knocking-down each gene in genome through RNA interference will reveal the genes that modulate the activity of dSREBP.Item Characterization of factors controling Germline Stem Cell Maintenance in Drosophila Melanogaster(2009-09-04) Park, Joseph Kwang; Mc Kearin, DennisDuring Drosophila oogenesis, a germline stem cell (GSC) divides asymmetrically to produce a renewed stem cell and a differentiated daughter cystoblast (CB) that will progress through the 14 stages of oogenesis to produce a mature egg. A myriad of factors regulate GSC maintenance; extrinsic signals from the somatic niche integrate with intrinsic GSC factors to control CB differentiation pathway. In particular, studies of a key differentiation factor, bam, underscored the important paradigm of maintaining GSC by preventing the initiation of the CB differentiation pathway. The Dpp/BMP signaling pathway from the GSC niche promotes quiescence of bam transcription in the GSC. In the differentiating CB, bam transcription is initiated and Bam protein, together with its protein partner Bgcn, functions to antagonize the translational repression mediated by Pumilio-Nanos (Pum-Nos) complexes. Repression of Pum-Nos activity by Bam-Bgcn complexes permits CB differentiation, presumably through the derepression of CB-promoting mRNAs. In this study, I investigated the transcription- and translation-dependent mechanisms controlling the GSC to CB transition. Through genome-wide expression profiling experiments, I showed that there are only minimal transcriptional differences between the GSC and CB. This supports previous studies that highlight the importance of translational repression in maintaining a GSC state. However, some of the transcriptional differences between the undifferentiated GSC and differentiated germ cells were uncovered by expression profiling experiments of germ cells lacking Stonewall (Stwl), a protein required for GSC maintenance through epigenetic regulation of CB-promoting genes. Data from gene profiling experiments of stwl bam and bam mutant germ cells suggest that a suite of genes is normally repressed by Stwl to maintain a GSC fate. In addition, I examined whether putative "stemness" genes identified from mammalian systems also affected Drosophila GSC fate using a pilot screen in a hypomorphic bam background. Components from the COP9 signalosome complex (CSN) and the SCF E3 ubiquitin ligase complex were identified as dominant Suppressors of bam, Su(bam). Since both the CSN and SCF complexes are involved in protein degradation, the suppression of the bam phenotype suggests that they may be involved in stabilizing Bam function through abrogation of Bam turnover. Indeed, though ubiquitinated Bam isoforms were not identified, the abundance of Bam protein was increased in Su(bam) heterozygous animals. In other studies, I examined the requirement for the microRNA pathway in GSC maintenance through the examination of the double-stranded RNA-binding protein Loquacious (Loqs). Loqs enhances Dicer-1's ability to process pre-miRNA hairpin moieties to mature miRNA duplexes. Loqs is required for viability and germline mosaic analysis of loqs GSCs indicates an intrinsic, cell-autonomous requirement for the miRNA pathway in GSC maintenance. Loqs localizes to putative RNP complexes and a specialized region of the oocyte cytoplasm, termed the pole plasm. These data suggest that Loqs is a component of protein-RNA complexes that may be involved in mRNA translational inhibition. In summary, my studies revealed that GSC maintenance is achieved through the repression of CB-promoting factors; epigenetically through the actions of Stwl and other histone-associated proteins and translationally through the actions of the miRNA pathway via Loqs and Dicer-1. My studies provide insights into the understanding of the CB-promoting factors that are held inactive in the GSC and suggest that other stem cell systems may similarly employ multiple layers of repressive mechanisms to maintain a stem cell state.Item Diazepam binding inhibitor and tolerance to ethanol in Drosophila melanogaster(2012-12) Robles, Roseanna Beth; Atkinson, Nigel (Nigel S.); Aldrich, Richard; Duvauchelle, Christine; Mihic, John; Zakon, HaroldTolerance to ethanol is an endophenotype of alcoholism, allowing the study of a complex psychiatric condition using animal models. To identify new genes involved in the acquisition of tolerance, I designed an automated and high-throughput tolerance assay and screened a collection of deficiency mutants for the inability to develop tolerance. The screen yielded several “regions of interest” where more than one overlapping deficiency failed to develop tolerance. One of these regions comprised nine genes, and testing the expression levels of each gene revealed that diazepam binding inhibitor (Dbi) showed grossly increased expression in the deficiency mutant compared to wild type. Another mutant stock, with a P-element transposon inserted downstream of the Dbi gene, both failed to develop tolerance and showed further increased expression of Dbi. There are two insulator binding sites flanking Dbi, and the P-element transposon also contains insulator binding sites. Based on these results, it was hypothesized that an insulator complex kept Dbi expression low in wild type flies and that disrupting the insulator complex allowed aberrantly high expression of Dbi in the mutants. Furthermore, we assumed that induction of Dbi blocked tolerance by making the mutants resistant prior to the first sedation. A UAS-DBI transgene was constructed to over-express Dbi. Induction of the UAS-DBI with a heat shock gal4 driver induced resistance to ethanol sedation; a similar response was observed in the parental control, but the effect was smaller. Although driving UAS-DBI with the neural elav-gal4 driver did not block tolerance, the experimental stock was resistant to ethanol sedation compared to the parental controls, indicating that increased Dbi expression produced “pre-tolerance.” To confirm the theory that insulator disruption was responsible for the increase in Dbi and the resulting no-tolerance phenotype, the P-element in the second mutant was mobilized by introducing a transposase source. These offspring lines were analyzed using qualitative PCR to determine whether the transposon excised precisely, left a portion of the transposon behind, or removed some of the flanking region. A precise excision mutant was identified, but this mutation did not rescue tolerance as predicted. This result might indicate that genetic background was the cause of the no-tolerance phenotype, or it might indicate that the excision was not exactly precise and removed the native insulator binding site, causing the insulator complex to remain disrupted.Item Fatty Acid Auxotrophy in Drosophila Larvae Lacking SREBP(2006-08-11) Kunte, Amit Sudhakar; Brown, MichaelA rapid increase in size is a major characteristic of larval development in Drosophila melanogaster. Such growth presumably requires the concomitant production of membrane lipids and is also accompanied by a significant accumulation of neutral lipid stores. Growing larvae must accumulate fatty acids to permit the synthesis of these lipids. Interestingly, wild type Drosophila can grow in the complete absence of exogenous fatty acids. This dissertation reports the finding that a lipogenic transcription factor, dSREBP (Drosophila Sterol Regulatory Element Binding Protein), is essential for the maintenance of this prototrophy. Drosophila larvae lacking dSREBP demonstrate a profound growth deficit in the second instar and die before reaching third instar. This is accompanied by transcriptional deficits in fatty acid synthetic genes. The growth deficit and lethality can be reversed by supplementing the culture medium with fatty acids. The most effective fatty acid, oleate, rescues 80 percent of dSREBP mutants to adulthood. Thus, a lack of dSREBP renders larvae auxotrophic for fatty acids. A reporter system demonstrates that dSREBP is active in tissues known to be involved in lipid metabolism- the fat body, oenocytes and anterior midgut. Finally, as expected of an end-product inhibited metabolic pathway, dSREBP activity can be suppressed by dietary supplementation with lipids. Thus, the dSREBP pathway coordinates endogenous synthesis with the dietary provision of exogenous lipids. These results establish Drosophila as a viable model for the genetic study of the SREBP pathway and provide the first evidence that, at an organismal level, the essential role of the pathway is the accumulation of lipids. The auxotrophic mutants and other reagents described here should be useful tools for further study of the SREBP pathway in particular and fatty acid metabolism in general.Item Feedback Regulation of Wnt Signaling By Naked Cuticle (Nkd) During Drosophila Embryogenesis(2008-05-12) Chan, Chih-Chiang; Wharton, Keith A.Wnt/beta -catenin signals are essential for many developmental and physiological processes in animals. Deregulation of the Wnt signaling pathway in mammals can cause diseases such as birth defects, cancer, osteoporosis, and diabetes. In Drosophila, the naked cuticle (nkd) gene antagonizes the Wnt/beta -catenin signaling in every segment of the embryo. Nkd is a modular, evolutionarily conserved protein that uses an EF-hand motif and adjacent sequences to target the cytoplasmic Wnt signal transducer Disheveled (Dsh). The mechanism by which Nkd antagonizes Wnt signaling in Drosophila embryos is not well understood. The abundance and bulk distribution of Dsh is not altered in nkd mutants as compared to wild type embryos, and overexpression of Nkd transgenes in nkd mutants did not alter Dsh distribution or abundance by confocal microscopy. Nkd transgenes lacking Dsh-binding regions were mostly able to rescue nkd mutants, suggesting that the Dsh-binding regions of Nkd contribute little to Nkd activity, at least when the transgenes were overexpressed. In this thesis, I have investigated non-Dsh binding regions that are critical for Nkd function. Our lab's findings indicate that a conserved 30 amino acid motif is essential for Nkd nuclear localization and function. Substitution of the 30aa motif with a heterologous nuclear localization sequence (NLS) rescued some nkd mutants to adulthood. In support of Nkd's role in the nucleus, Nkd binds to Importin-alpha 3, an adaptor for the canonical nuclear import apparatus. I identified that Nkd associates with Importin-alpha 3 via a motif ("D6") that is conserved between D. melanogaster and D. pseudoobscura. NkdΔD6, lacking the Importin-alpha 3-binding motif, was defective in nuclear localization and in rescuing nkd mutants. RNAi knockdown of importin-alpha 3 prevented the nuclear localization of Nkd. The findings that Nkd possesses two NLSs, each of which is required for function, and that Nkd associates with a component of the nuclear import apparatus, suggest that Nkd antagonizes the Wnt/beta -catenin signaling in the nucleus. Furthermore, I also addressed the function of the N-terminus of Drosophila Nkd. Unlike mammalian Nkd homologs that have N-terminal myristoylation consensus sequences responsible for membrane association, the N-terminus of Drosophila Nkd, also conserved in mosquito Nkd, lack such a sequence. Nonetheless, Nkd's N-terminus was required for function and membrane association. Substitution of the N-terminus with heterologous myristoylation sequences did not restore nkd function, indicating that the mechanism by which Drosophila Nkd associates with the membrane is different than mammalian Nkds. Therefore, Nkd appears to function in the membrane, in the cytoplasm to target Dsh, and in the nucleus to antagonize Wg signaling.Item Functional characterization of the role of Bruno protein in translational regulation and germ line development in Drosophila melanogaster(2006-05) Yan, Nan, 1979-; Macdonald, Paul M.Both body axes of the Drosophila egg are determined by localization of several mRNAs to specific regions within the oocyte. One of these mRNAs, oskar (osk), is required for posterior body patterning. Localization and translational control are both crucial for the correct deployment of osk. Bruno (Bru) binds specifically to the 3’UTR of the osk mRNA and represses osk translation. In this dissertation, I first describe a genetic screen looking for dominant modifiers of the arrest (aret) mutant phenotype (aret encodes Bru). Two modifiers suggested additional targets for Bru action. One is Star, a gene that contributes to provision of Gurken activity. The second suggested target is a gene acting in the Delta signaling pathway. A final modifier, Lk6, encodes a protein kinase predicted to regulate eIF4E. I also took a biochemical approach trying to understand how Bru regulates osk translation. Bru protein contains three RNA Recognition Motifs, but the remainder of the protein had no known function. I identified a domain, which is required for interaction to Bru itself, Cup and Apontic. Subsequent analysis of mutant forms of Bruno defective in these interactions led us to an unexpected discovery that Bru also acts as an activator of osk translation. Parallel analysis of Bru binding sites in osk 3’UTR fully support the notion that Bru has a dual role. There are two clusters of Bru Recognition Elements in either end of osk 3’UTR. Point mutations in one cluster cause overproduction of Osk protein while point mutations in the other cluster largely prevent translation of the message. To understand the molecular basis of the opposing roles of Bru, I used quantitative methods to better define and compare the binding of Bru to the different regulatory elements: those that either repress or activate osk mRNA translation. Using purified components I found that Bru binds to two clusters of binding sites in the osk 3’UTR differently, in terms of affinity, cooperativity and apparent compaction of the RNA. This work raises the possibility that the details of how Bru binds its substrate may determine whether it acts as a repressor or an activator.Item Genetic Dissection of Heart Development in the Fruit Fly Drosophila Melanogaster(2007-12-18) Yi, Peng; Olson, Eric N.The early morphogenetic mechanisms involved in heart formation are evolutionarily conserved. The Drosophila heart, known as the dorsal vessel, functions as a pulsatile tube-like organ containing an inner layer of contractile cardial cells that adhere tightly to an adjacent layer of pericardial cells. A genetic screen for genes that control Drosophila heart development revealed a cardiac defect in which pericardial and cardial cells dissociate causing loss of cardiac function and embryonic lethality. This phenotype resulted from mutations in the genes encoding HMG-CoA Reductase, downstream enzymes in the mevalonate pathway, and G-protein Ggamma 1, which is geranylgeranylated, thus representing an endpoint of isoprenoid biosynthesis. These findings reveal a cardial cell-autonomous requirement of Ggamma 1 geranylgeranylation for heart formation and suggest the involvement of the mevalonate pathway in congenital heart disease. In addition, we found that the heterotrimeric G proteins Gbeta 13F and G-oalpha 47A together with the RGS (regulator of G protein signaling) protein Loco function in the same pathway as Ggamma 1 to regulate septate junction formation in cardial cells of the Drosophila heart. We also present evidence that the septate junction protein Sinuous interacts with Pericardin, a matrix protein secreted by pericardial cells, providing the molecular basis for cardial-pericardial cell adhesion and serving as a mediator of the actions of the mevalonate pathway and heterotrimeric G protein signaling in Drosophila heart development.Item Illuminating the P53 Regulatory Network in Genetic Models(2011-02-01T19:36:06Z) Lu, Wan-Jin; Abrams, JohnThe tumor suppressor gene p53 is mutated in more than 50% of human cancers, and functions as a central component of stress response machinery that mediates a wide variety of downstream responses. Interestingly, the evolutionary appearance of p53 preceded its role in tumor suppression, suggesting that there may be unappreciated functions for this protein. In order to examine physiologic functions of p53 in vivo, a green fluorescent protein (GFP) reporter was designed to follow the activation of this regulatory network in a genetic model, Drosophila melanogaster. By following the reporter during Drosophila development, physiological activation of the p53 regulatory network in the female germ line was discovered. It is provoked by the first enzymatic step for meiotic recombination and conserved in both flies and mice. The functional relevance of the p53 activities in the germ line was shown by the meiotic recombination frequency and genetic interactions with a meiotic effector gene, Rad54. Additionally, genotoxic stress selectively activates p53 in germ line stem cells and promotes regeneration of fertility after IR. Activation of p53 was also found in uncontrolled growth of germ cells by blocked differentiation, and surprisingly by overexpression of oncogenic protein in the germ line. Together, my thesis work indicate that the need for controlling growth by the p53 regulatory network is an evolutionary conserved feature, which may serve as a selective pressure to preserve this network. Future studies on the mechanisms of p53 actvities during meiosis and in response to oncogene activation could provide novel insights on its cancer-related functions.Item Mechanisms of Volatile Odorant Detection and Corresponding Behavioral Effects in Drosophila(2011-08-26T17:34:37Z) Ronderos, David Samuel; Smith, Dean P.One of the central problems in neuroscience concerns the molecular basis of behavior. Animals generate appropriate behavioral responses to environmental stimuli, which are mediated by primary sensory neurons of different modalities including sight, hearing, touch, taste, and smell. Olfaction serves a major role in conveying important information concerning a variety of behavioral determinants such as food sources, danger, and cues from other members of the same species. Drosophila melanogaster provides a tractable model in which to study both the mechanisms of olfaction and subsequent behavioral outputs based on olfactory input. Additionally, basic knowledge pertaining to insect olfaction provides valuable insights into potential targets that may be used to influence insect vectors of diseases (e.g., mosquitoes and malaria or dengue fever), or to improve agriculture by affecting pollination by foraging insects (e.g., honeybees). My research has focused on the molecular mechanisms involved in detection of pheromones, naturally-occurring insect repellents, and food source-derived odorants by primary sensory neurons in Drosophila and the corresponding behaviors generated by these stimuli. To date, the only known volatile pheromone in Drosophila is the male-specific hydrocarbon 11-cis-vaccenyl acetate (cVA). I took a genetic approach to study the role of cVA-induced neuronal activation, and found that activation of the at1 neuronal circuit involved in cVA detection, using a dominant-active form of the odorant binding protein LUSH, is both necessary and sufficient to induce sexually dimorphic mating behavior in Drosophila. In a separate study, I identified novel olfactory sensory neurons that detect the naturally-occurring insect repellent citronellal via a mechanism involving the ion channel TRPA1. Finally, I characterized the first odorant receptor shown to be expressed in neurons of the intermediate class of olfactory sensilla, Or83c, and found that it was highly specific for a sesquiterpene found in citrus fruit peel called farnesol (FOH). Furthermore, FOH detection by Or83c instructs oviposition preference in Drosophila via ai2a sensory neurons. These studies advance general knowledge of the architecture and complexity of the Drosophila olfactory system and provide insights into the molecular basis of behaviors in insects.Item Novel roles of the proteins Oskar and Bluestreak in germ cell formation and migration(2007) Jones, Jennifer Rebecca, 1978-; Macdonald, Paul M.The formation of germ cells in Drosophila melanogaster is dependent on the presence of ribonucleoprotein complexes called polar granules. A key component of these complexes is Oskar, a novel protein which has been shown to nucleate the granules. To investigate whether Oskar plays a further role in polar granule formation, I cloned the oskar gene from D. immigrans flies (osk[superscript imm]) and introduced it into D. melanogaster flies using P-element transformation. I found that osk[superscript imm] was able to rescue both the posterior patterning and germ cell formation defects of embryos from oskar mutant mothers. In addition, I found that the polar granules of embryos containing only Osk[superscript imm] as a source of Oskar protein resemble those found in D. immigrans embryos, indicating a new role for Oskar in determining the morphology of the polar granules. Germ cell formation in Drosophila is succeeded by migration of the germ cells to the site of gonad formation. A second line of research presented in this dissertation describes analysis of a novel protein important for both germ cell formation and migration, Bluestreak (Blue). Embryos from either heterozygous or homozygous Blue-mothers display defects in germ cell number and shape. I found that the ovaries of Blue-females have defects in the localization of Staufen and Oskar, sufficient to cause a reduction in pole cell number in embryos. In addition, genetic analysis of the interaction between Bluestreak and mutants which affect pole cell migration implicates Bluestreak in this process. Finally, I found that Blue localizes to centrosomes along with [gamma]-tubulin throughout the embryo, and to the nuclear membrane in pole cells. My findings introduce the possibility that Bluestreak may act to regulate germ cell migration in Drosophila.Item Relative viability of Drosophila melanogaster heterozygous for recessive lethals(Texas Tech University, 1967-06) Huang, Shiu LanNot availableItem The Role of a Neuron-Specific V-Atpase in Synapse Specification, Function, and Maintenance(2012-08-13) Williamson, Wallace Ryan; Hiesigner, Peter RobinThe Vacuolar-type (V-) adenosine triphosphatase (ATPase) is a proton-pumping nanomachine consisting of two multi-subunit, reversibly associating protein sectors, the cytosolic V1 sector and the membrane-bound Vo sector. The V1/Vo holoenzyme hydrolyses ATP to translocate protons across biological membranes thereby modulating lumenal and extracellular pH. Additionally, accumulating evidence suggests that the Vo sector has a role in membrane fusion when dissociated from the V1 sector, its proton-pumping partner. Published evidence for this includes a null allele for the neuron-specific a subunit in the Drosophila Vo sector, v100, which leads to defects in synaptic function that are unrelated to pH regulation. My project emerged from the need to explain why v100 has two additional phenotypes that are absent in other synaptic function mutants: functional and structural degeneration in photoreceptor cells and patterning defects in the visual system neuropil. I proposed that v100 has a previously undocumented role on a neuron-specific endo/lysosomal pathway in addition to its documented role in neurotransmitter secretion. To test my hypothesis in the context of only one of the two purported v100 functions, I generated transgenic animals with v100 mutations designed to specifically disrupt either acidification or membrane fusion. Using these genetic tools, I discovered that v100 has an essential role in sorting cargo into an endo-lysosomal pathway that concomitantly requires v100 for the acidification-dependent maturation of degradation-competent organelles. This 'sort-and-degrade' mechanism for v100 defines a neuron-specific degradation pathway that is required for synaptic specification, function, and maintenance. In developmental stages, v100 is required to 'sort-and-degrade' guidance receptors as part of the synapse specification program. In the adult, the 'sort-and-degrade' mechanism provides additional degradative capacity to neurons, a cell type that must often maintain homeostasis for unusually long periods of time. Finally, I provide evidence that the role for V100 in membrane fusion requires a direct, physical interaction with Syntaxin-1, an interaction that can be specifically disrupted in vitro and in vivo. In brief, my results provide mechanistic insight into the acidification-independent role of v100 and reveal the existence of a neuron-specific endo-lysosomal pathway on which v100 functions to 'sort-and-degrade' cargo in order to meet the special needs of a neuron in development, function, and maintenance. [Keywords: Drosophila, intracellular trafficking, development, degeneration, lysosomal, endosomal, degradation, acidification]Item The Role of Double-Stranded RNA Binding Protein R2D2 and Lump in Drosophila Gametogenesis(2010-05-14) Sanders, Charcacia Tiana; Smith, DeanIn Drosophila melanogaster, fertility requires the successful development of germline cells into mature gametes. This process is dependent on multiple factors that coordinate migration, proliferation, and differentiation of germline stem cells. Previous studies have shown dsRNA binding proteins have an important role in the induction and maintenance of germ cells. In this study, I investigated the requirement of dsRNA binding proteins, R2D2 and LUMP, in Drosophila melanogaster gametogenesis. I show R2D2 functions in the ovary, specifically in the somatic tissues giving rise to the stalk and other follicle cells critical for establishing the cellular architecture of the oocyte. Most interestingly, the female fertility defects in r2d21 are dramatically enhanced when one copy of the dcr-1 gene is missing. This indicates dicer-1 and r2d21 operate in the same fertility pathway. Furthermore, Dicer-1 protein coimmunoprecipitates with R2D2 antisera, indicating these proteins function in the same protein complex. Thus, r2d21 mutants have reduced viability and defective female fertility that stems from abnormal follicle cell function and Dicer-1 impacts this process. This is the first indication R2D2 functions beyond its known role in RNA interference to include ovarian development in Drosophila. I also studied a second putative double-stranded RNA binding protein (dsRBP). I found that lump is required for male fertility and there is an absence of motile sperm in lump mutant testes squash preparations and the seminal vesicles. The early stages of spermatogenesis, including mitosis, meiosis, and cytokinesis steps are unaffected in lump mutants. This indicated lump is likely required late in sperm development. The spermatid individualization complex is disrupted consistent with an individualization-deficient phenotype. A wildtype genomic rescuing transgene was able to rescue fertility and individualization. However, a lump transgene carrying a point mutation in the first dsRNA binding domain did not rescue fertility or individualization suggesting this domain is essential for lump function. Thus, it is likely that these two dsRNA binding proteins regulate gene expression in the ovary and testes and are essential for normal fertility.Item Studies of the Hippo Signaling Pathway(2012-08-13) Yue, Tao; Jiang, JinHow multicellular organisms control their growth to reach proper organ size during development is a fascinating question. Recent studies, initially from Drosophila, have identified the Hpo tumor suppressor pathway as a crucial mechanism that controls tissue growth by inhibiting cell growth, proliferation and survival. Deregulation of the Hpo pathway has been implicated in various human cancers. Central to the Hpo pathway is a kinase cassette consisting of four tumor suppressor proteins, the Ste20-like kinase Hpo, the WW domain-containing protein Salvador (Sav), the NDR family kinase Warts (Wts) and the Mob family protein Mats. The kinase activities of Hpo and Wts are facilitated by their regulatory proteins Sav and Mats, respectively. Activated Hpo/Sav complex phosphorylates and activates the Wts/Mats complex, which in turn phosphorylates and inactivates the transcriptional coactivator Yorkie (Yki). Phosphorylation of Yki restricts its nuclear localization through recruiting 14-3-3. When the activity of the Hpo/Wts kinase cassette is compromised, Yki forms complexes with transcription factors including Scalloped (Sd) and translocates to the nucleus to activate Hpo pathway target genes, including cyclin E, diap1, and the microRNA bantam that regulate cell growth, proliferation and survival. To identify novel components of the Hpo signaling pathway, I carried out a genetic modifier screen in which flies carrying GMR-Gal4 and UAS-Yki were crossed to a collection of transgenic RNAi lines from Vienna Drosophila RNAi center (VDRC) and Bloomington stock center, and looked for enhancers or suppressors of the overgrown eye phenotype caused by Yki overexpression. Through this screen, I have found that Echinoid (Ed), an immunoglobulin domain-containing cell adhesion molecule, acts as an upstream regulator of the Hpo pathway. Loss of Ed compromises Yki phosphorylation, resulting in elevated Yki activity that drives Hpo target gene expression and tissue overgrowth. Ed physically interacts with and stabilizes the Hpo-binding partner Sav at adherens junctions. Ed/Sav interaction is promoted by cell-cell contact and requires dimerization of Ed cytoplasmic domain. Overexpression of Sav or dimerized Ed cytoplasmic domain suppressed loss-of-Ed phenotypes. I propose that Ed may link cell-cell contact to Hpo signaling through binding and stabilizing Sav, thus modulating the Hpo kinase activity. Furthermore, the Cul4/WDR40A complex has also been identified as a genetic modifier for the Hippo signaling pathway. However, the exact mechanism by which this complex regulates the Hippo signaling pathway need to be further addressed. [Keywords: hippo pathway, echinoid, Salvado, cell adhesion, cell contact]