Anatomy and Neuroscience - Theses
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ItemThe adult Drosophila salivary gland: developing a new epithelial research model( 2021)Arthropod-borne viruses, also known as arboviruses, are transmitted to humans through arthropod bites. Viruses such as Dengue, West Nile, and Zika are transmitted through mosquito bites and cause serious illness in humans. These viruses are injected into a human host in the saliva of a feeding mosquito, a process that hinges on the virus invading the mosquito’s salivary glands. Therefore, a deep understanding of insect salivary glands is an important step in learning how to control arboviruses. One of the world’s most popular research organisms, Drosophila melanogaster, is a relative of the mosquito and of other insect disease vectors. Drosophila salivary glands could provide an excellent model for studying the transmission of arboviruses, unfortunately extraordinarily little is known about the glands of adult Drosophila. The aim of this research project was to develop the adult Drosophila salivary glands as a research model for studying the interactions between arboviruses and insect salivary glands. Since little is known about the glands, my investigations focused on understanding the structure, function, and maintenance of the cells within the salivary gland. To understand the structure of salivary glands, I first investigated the structure of the organ, before looking closely at individual cells. I characterised the structure of the cells by investigating localisation of cell-junctions, cytoskeletal elements, and cell-polarity markers. I also observed the establishment of these morphological features throughout different stages of development. Second, by combining the structural data with investigations into intracellular signals and membrane channels, I provided a hypothesis of the functions of salivary gland cells. Then, by analysing cell division and cell-maintenance pathways in the salivary glands, I provided an insight to how the salivary gland cell population is maintained. From this project the salivary glands emerged as a multifaceted research model that could be used to investigate arboviral diseases, epithelial tissues, and amitotic division.
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ItemRibosomal protein depletion in the Drosophila haematopoietic compartment alters cell fate determination( 2020)Ribosomes are essential components of the translational machinery, required for cells to effectively double their protein content in order to undergo cell division. Despite the obvious need for ribosomes, due to the high energetic cost, ribosome biogenesis is tightly regulated by numerous pathways in response to cellular and extracellular signalling cues. Predictably, dysregulation of ribosome levels strongly correlates with disease. Increased ribosome biogenesis is observed in many cancers and decreased ribosome biogenesis underlies a class of developmental disorders collectively termed ribosomopathies. Somewhat surprisingly, given the ubiquitous need for ribosomes, loss of ribosomal proteins has also been associated with tissue overgrowth in both human disease and model organisms, including zebrafish and Drosophila. In the latter, reported mechanisms for overgrowth due to global reduction of ribosomal proteins comprise extrinsic effects, whereby a smaller hormone-secreting gland delays animals in their growth phase to result in larger wings and eyes. Many ribosomopathies are associated with lineage-specific defects, with ribosomal protein loss linked with haematopoietic compartment pathologies, particularly lineage depletion and anaemia. Although lineage depletion phenotypes are well established to arise from apoptosis induced by nucleolar stress, there is also an increased incidence of cancer associated with these ribosomopathies for which mechanisms remain unclear. This thesis has taken advantage of the powerful genetic tools available in Drosophila to achieve tissue-specific depletion, in the larval hematopoietic compartment (the lymph gland), of the two ribosomal proteins most commonly lost in the ribosomopathy Diamond Blackfan Anaemia (DBA), S19a (RpS19a) or S24 (RpS24). In contrast to the haematopoietic lineage depletion observed in human disease, we report cell-intrinsic overproliferation and tissue overgrowth following depletion of either RP. However, despite gross phenotypic similarities, depletion of RpS19a and RpS24 resulted in distinct cell death and differentiation defects. Moreover, while RpS24 resulted in the expected decrease in mature ribosomes, RpS19a depletion resulted in an inexplicable increase in ribosomes. Transcriptome and proteome analyses of RP-depleted lymph glands revealed upregulation of metabolic and signalling pathways; however, factors involved in transcription and translation were disproportionately increased at the protein level, consistent with altered translation. Although certain proteins were elevated after knockdown of either RP, some were specifically increased by RpS19a or RpS24 depletion. Of the 8 candidates tested for capacity of co-depletion to suppress overgrowth defects only 5 (STAT, TOP1, Osa, TCTP and Cdk12) suppressed the RpS24 lymph gland phenotype, suggesting the requirement for increased abundance of these factors in overgrowth. In the case of RpS19a, although depletion of dHEATR1 and TCTP supressed overgrowth, only depletion of Osa, a key component of the SWI/SNF chromatin remodelling complex, suppressed overgrowth through a restoration of progenitors. The increased Osa observed in RpS19a and RpS24 knockdown is, therefore, required for progenitor depletion and lymph gland overgrowth. Further studies are required to determine whether differential translation of Osa, and the other candidates, contributes to lymph gland overgrowth due to depletion of RpS19a or RpS24.
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ItemSuppressors of oncogenic Cbl in the Drosophila eye.( 2018)Cbl is an E3 ligase, and downregulates several cellular signalling pathways, in this role by targeting receptor tyrosine kinases for endocytosis. Mammalian Cbl was first identified as the full-length isoform of v-Cbl, a C-terminal truncated dominant negative oncogene that permits binding of v-Cbl to Cbl targets but does not facilitate their ubiquitination. This results in constitutive activation of the receptor tyrosine kinase. In this thesis, I used a Drosophila analogue of v-Cbl, named Dv-cbl. GMR>Dv-cbl had been used prior to the commencement of this study to screen for modifiers of its rough and overgrown eye phenotype using the Gene search system, a transposon-based inducible expression system. In this study, a subset of the suppressors of the GMR>Dvcbl phenotype from that screen, and two other representative lines were further investigated. The published interactions and functions of the genes implicated by the GS lines are discussed and a method of suppression of the GMR>Dv-cbl phenotype by each line is suggested. In the published work presented in this thesis, the Akap200 expressing lines EP2254 and GS2208 were further studied. Expression of Akap200 in EP2254 was confirmed via mRNA in situ hybridisation, and its ability to also suppress the Ras85DV12 phenotype was confirmed. The ability of EP2254 to suppress GMR-Dv-cbl and sev-Ras85DV12 coexpression was confirmed. When GMR-Dv-cbl and sev-Ras85DV12 are coexpressed, a phenotype that is greater than the cumulative phenotype of each would suggest arises. In fact, GMR-Dvcbl (where Dv-cbl was directly driven from the GMR promoter) was used instead of GMR-Gal4, UAS-Dv-cbl (GMR>Dv-cbl) as the coexpression of GMR>Dv-cbl and sev- Ras85DV12 results in lethality, and coexpression of GMR-Dv-cbl and sev-Ras85DV12 did not. Alone, each has a mildly rough eye. I showed that EP2254 was able to suppress this phenotype and that this suppression was partially independent of apoptosis. The endogenous function of Akap200 in the Drosophila eye was then investigated. An mRNA in situ hybridisation experiment showed that endogenous Akap200 is present in the eye disc, and a series of immunohistochemical stains showed that Akap200 was expressed in a subset of photoreceptor cells. Knockdown of Akap200 using RNAi lines showed that endogenous Akap200 was having a modifying effect on the GMR>Dv-cbl phenotype, as knockdown of Akap200 enhances the GMR>Dv-cbl phenotype. A recent study suggests that Notch is protected from internalisation by Cbl by Akap200, which is consistent with the results in this thesis.
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ItemSnail - an important regulator of Drosophila GSC homeostasis( 2016)The Drosophila testis is a long coiled tube-like structure with male germline stem cells (GSCs) residing in the apical tip. 8-10 GSCs are arranged in a rosette shape around a group of somatic cells termed the hub, which secretes factors that regulate stem cell self-renewal. Due to asymmetrical division of GSCs, one daughter cell remains in direct contact with the hub and therefore maintains stem cell identity, while the other is displaced from the hub and commences differentiation to become a gonialblast. The gonialblast undergoes four rounds of mitotic divisions with incomplete cytokinesis, resulting in a cyst of sixteen interconnected spermatogonia which then progress through pre-meiotic S-phase and differentiate to become spermatocytes. There is a delicate balance between self-renewal of GSCs and differentiation of germ cells, which is required for tissue maintenance and regeneration. Snail proteins are zinc-finger DNA-binding proteins that act as transcriptional repressors of target genes via the binding to specific sequences termed ‘E-boxes’ in the promoter sequence. Drosophila has three Snail members which include: Snail, Escargot and Worniu, while the mammalian Snail family is comprised of Snai1/Snail, Snai2/Slug and Snai3/smuc. Snail proteins play a crucial role in initiating epithelial to mesenchymal transitions (EMT), both in normal physiology and disease. This project reveals an important role for the transcription factor Snail in the regulation of GSC maintenance. I have utilised a dominant negative transgene of escargot (esgDN) in order to block the function of all three Snail family members, which resulted in a severe GSC loss phenotype. Only the expression of a WT snail transgene was able to partially rescue the phenotype. Despite its high expression in somatic cells and spermatogonia, loss of Escargot function in GSCs has no effect. Conversely, I showed that snail is expressed in very low levels in the germline but snail mutant GSCs are lost from the niche. My results demonstrate that the loss of GSCs is not due to cell death or misregulated differentiation, but due to a disruption in adhesion of GSCs to the hub, which ultimately results in loss of GSC identity. Snail proteins were previously shown to have an important role in mammalian gonads, thus suggesting a conserved role for Snail in regulating spermatogenesis.
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ItemFine-tuning expression of dMYC: how the single stranded DNA binding proteins Hfp and Psi regulate dMYC expression in Drosophila( 2015)MYC proteins are critical regulators of growth and cell cycle progression essential to animal development (Lee and Quinn, 2014; Lee et al., 2012; Levens, 2010). MYC is overexpressed in approximately 70% of all cancers (Dang, 2010; 2014). Understanding the molecular mechanisms that control MYC activity is crucial to understand normal development and may lead to insights into MYC dysregulation in diseases such as cancer. This thesis investigates the regulation of MYC abundance at the transcriptional level. In all multicellular animals, cells and tissues must have the capacity to quickly respond to environmental and cellular signaling pathways to coordinately regulate MYC transcription and cell growth. Based on in vitro studies we predicted that the presence of paused, but transcriptionally active, RNA polymerase II (RNA Pol II) on the MYC promoter is required for a rapid response to cell signaling. Of particular interest to this project, David Levens' ex vivo cell culture studies have shown that in response to mitogenic signals in serum the single stranded DNA binding protein Far upstream element Binding Protein (FBP) is enriched on the promoter of the MYC oncogene. FBP binding is associated with depletion of RNA Pol II from the promoter and increased MYC transcription (Liu et al., 2006). Subsequent to the peak in MYC mRNA, FBP Interacting Repressor (FIR) is detected at the FUSE and is required for the return of MYC transcription to basal levels. Moreover, the FIR protein is required for repression of MYC transcription via interaction with the general transcription factor (TFIIH) complex (Liu et al., 2006; 2001), and is dysregulated in human colorectal cancer (Matsushita et al., 2006). This mechanism of MYC repression appears to be conserved between mammals and Drosophila, as we have demonstrated that the homolog of FIR (Hfp) represses Drosophila MYC (dMYC) in a manner dependent on the XPB helicase subunit of the TFIIH complex (Mitchell et al., 2010). Xeroderma Pigmentosum (XP) is a rare autosomal-recessive disease characterized by pigment changes, premature ageing and in a few patients malignant tumour development (Cleaver, 2005; Oh et al., 2006). Moreover, cancer progression occurs in certain patients, but not others, with identical C terminal mutations in the XPB helicase (Oh et al., 2006). The issues of compound heterozygosity and mixed genetic backgrounds have compounded problems of resolving the disease mechanism in the small number of XPB patients. Thus mechanisms driving overproliferation and cancer associated with these XPB mutations are currently unknown (Cleaver, 2005). With Drosophila we have used single alleles and, for the first time, made clear connections between genotype and phenotype. Using Drosophila models we show that C-terminally truncated XPB/Hay alleles enhance tissue overgrowth due to reduced abundance of FIR/Hfp, to further impair transcriptionally repression of dMYC. Thus, we predict defective transcriptional repression of MYC by FIR/Hfp might provide one mechanism for spontaneous cancer progression in XP patients. Dissecting the role of the FBP protein in MYC regulation has been complicated by the fact that mammals have multiple FBP family members (FBP 1, 2 and 3), which bind highly overlapping transcriptional targets (Chung et al., 2006). Originally, based on the finding that FBP is required for activated MYC transcription in vitro (He et al., 2000; Liu et al., 2001), we predicted FBP might play a role in activating RNA pol II release and MYC transcript elongation. We used Drosophila models to test whether the sole FBP family member, Psi, is required for dMYC transcription in vivo. Here we demonstrate that depletion of FBP/Psi reduced dMYC mRNA levels, suggesting FBP/Psi is normally required for maintaining endogenous levels of dMYC. Moreover, ChIP for poised RNA Pol II (Ser 5) proximal to the dMYC transcription start site revealed that FBP/Psi depletion reduced RNA Pol II Ser 5 enrichment. Together these data suggest FBP/Psi is required for maintaining poised RNA Pol II on the dMYC promoter and for maintaining dMYC transcription in vivo. Based on David Levens' extensive in vitro studies, we predict that FBP/Psi and FIR/Hfp are required for “fine-tuning” signal stimulated MYC transcription. We conducted a candidate screen of known mitogenic signals to identify those capable of most strongly stimulating dMYC transcription in vivo. The Ras/ERK pathway was the most robust activator of the dMYC promoter, and was able to significantly increase dMYC mRNA abundance. Importantly, we demonstrated that Ras-stimulated dMYC transcription was significantly reduced by depletion of FBP/Psi. Thus, endogenous levels of FBP/Psi are required for maximal stimulation of dMYC transcription by mitogenic signals in vivo.
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ItemFlamingo/Starry Night in embryonic abdominal sensory axon development of Drosophila( 2008)The seven-pass transmembrane atypical cadherin, Flamingo (also known as Starry Night) is evolutionally conserved in both structure and function in vertebrates and invertebrates. It plays important roles during the establishment of planar cell polarity (PCP) of epithelial tissues and during the development of axons and dendrites in both peripheral and central neurons. This thesis looks at the role of Flamingo/Starry Night in axon growth and guidance in the embryonic abdominal peripheral nervous system (PNS) of Drosophila. It describes the expression pattern of Flamingo in the PNS and its environment. A combination of single cell labelling and immunohistochemical techniques was used to define the effect of mutations in flamingo as well as several genes coding for potential Flamingo interaction partners. Rescue- and over-/mis-expression experiments featuring targeted expression of either a wild type version or mutant versions of flamingo provide information on the cellular and molecular mechanisms by which Flamingo regulates sensory axon development. Loss of Flamingo function results in a highly penetrant axon stall phenotype. Both sensory and motor axons frequently halt their advance early along their normal trajectories. Flamingo appears to mediate an axon growth promoting signal upon contact of sensory growth cones with specific early intermediate targets. Expression of Flamingo in sensory neurons is sufficient to rescue the mutant sensory axon phenotype. This rescue is at least partially independent of most of the extracellular region of the Flamingo protein. While Flamingo was previously found to have homophilic adhesion properties in vitro and appears to function by a homophilic mechanism during the neurite development of several types of neurons, this study supports a heterophilic signalling mechanism by which Flamingo fulfils its role in abdominal sensory axon growth promotion.