Vanessa Auld

Peripheral nervous system (PNS) health is largely dependent on proper glial cell functioning during development. Myelinating and non-myelinating Schwann cells are glial cells in the PNS that ensheathe and protect axons. Communication between Schwann cells and the extracellular matrix (ECM) is essential for PNS development. The ECM protein laminin, and its receptor Dystroglycan [Dg; part of the Dystrophin-glycoprotein complex (DGC)], are important for myelinating Schwann cell development, however little is known about the mechanisms underlying the role of laminins and Dg/DGC in non-myelinating Schwann cell development. We use developing Drosophila wrapping glia (WG), which ensheathe axons similarly to non-myelinating Schwann cells, as a model to study the role of laminin/Dg in non-myelinating Schwann cell development. We found strong expression of LanA (one of two laminin alpha subunits in Drosophila), around WG. Wing blister, the other laminin alpha subunit, is not strongly expressed in the peripheral nerve, indicating that the LamininA isoform is the primary laminin isoform expressed here. We found that WG express LanA, and knockdown of LanA in WG caused perinuclear membrane accumulations and a reduction in WG-axon contact. We found that LanA is deposited in a polarized manner—LanA is preferentially localized between WG and axons (rather than between WG and its adjacent glial layer, subperineurial glia), and preferentially around motor axons versus sensory axons. We also found the laminin receptor Dg is expressed on WG membranes, and we identified differential expression of Dg isoforms in different layers of the peripheral nerve. Knockdown of Dg in WG causes a WG morphology defect, reduction in WG-axon contact, and reduction in laminin deposition in the WG-axon area. We also found that knockdown of Dystrophin, the intracellular binding partner to Dg, phenocopies the Dg-mediated WG morphology defect, suggesting that these proteins likely function together in mediating WG morphology during development. Overall, we characterized a novel localization pattern and function of laminin and Dg in Drosophila WG during development. Due to the highly conserved nature of laminins and DGC proteins, our results have implications for non-myelinating Schwann cell development—thus improving our understanding of the factors underlying PNS development in all animals.

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Development and maintenance of the peripheral nervous system (PNS) relies on glial cells that insulate and protect axons. In invertebrates, wrapping glia isolate axons into separate bundles similar to non-myelinating Schwann cells (NMSCs) in vertebrate Remak bundles. The mechanisms by which NMSCs communicate with each other remains unknown. In this thesis, we focused on identifying and characterizing proteins required for glia-glia communication in non-myelinating classes of glia using the Drosophila peripheral nerve as our model. We show that Innexin1 (Inx1) and Innexin2 (Inx2) based gap junctions (GJs) exist between two peripheral glial layers, the subperineurial glia (SPG) and wrapping glia (WG). WG survival is dependent on its communication with the SPG and is mediated by a channel rather than adhesive function of Inx2. Inx2 GJs mediate calcium pulses exclusively in the SPG and WG survival is not dependent on Ca²⁺ and inositol 1,4,5-trisphosphate (IP3). Therefore, we find that GJs mediate glia-glia communication to ensure the survival of WG through an unknown mechanism.We next tested the role of scaffolding complexes in mediating glia-glia communication and screened for the role of the PSD95-Dlg-ZO1 (PDZ) family of proteins. We identified a role for Dlg5, a membrane-associated guanyl kinase protein, in peripheral glia. Loss of Dlg5 results in glial disruptions, including loss of septate junction formation and axonal ensheathment. Dlg5 has multiple roles identified in other systems including trafficking of cadherins. However, in glia the loss of Dlg5 did not affect cadherin localization to spot adherens junctions (SAJs). Therefore, we find that Dlg5 plays a novel role in peripheral glial development. SAJs were previously identified in the Drosophila peripheral glia, but the composition and function of this complex had not been characterized. We find that classical cadherins associate with catenins in the peripheral nerve, and loss of DE-Cad but not DN-Cad leads to disruptions in glial morphology. However, loss of DE-Cad does not affect SAJ assembly, suggesting a redundancy with DN-Cad in peripheral glia.Taken together this thesis provides novel insights and proposed models by which glia communicate in the PNS and will help direct future work in NMSCs in all animals.

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This thesis investigates the kinase-mediated regulation of the tricellular junction protein, Gliotactin and signaling pathways involved in Gliotactin overexpression-induced detrimental phenotypes. Tricellular junctions (TCJ) are uniquely placed permeability barriers formed in polarized epithelia where tight junctions in vertebrates or septate junctions in invertebrates from three cells converge. Misregulation of TCJ specific proteins is detrimental to life. However, mechanisms of their localization, maintenance, and potential signaling are largely unknown. Gliotactin is a transmembrane protein unique to TCJ in Drosophila and is essential for the maturation and maintenance of both bicellular and tricellular septate junctions. However, overexpression of Gliotactin leads to the spread of Gliotactin away from the TCJ and disrupts epithelial architecture by signaling for overproliferation, delamination, migration and apoptosis. One mechanism to control Gliotactin is phosphorylation of two highly conserved tyrosine residues and subsequent endocytosis. However, Gliotactin tyrosine phosphorylation also elicits detrimental phenotypes when dysregulated. The kinases involved in Gliotactin phosphorylation had not been broadly investigated prior to this work. We carried out an RNAi screen for phospho-regulators (kinases and some kinase-associated proteins) to determine which could modify the detrimental phenotypes triggered by Gliotactin overexpression. Four suppressors, four partial suppressors, and 53 enhancers were identified by screening 275 RNAi lines covering 164 genes. We determined that Gliotactin overexpression phenotypes involved TNF-JNK, PI3K-Akt signaling pathways and Btk29A. C-terminal Src kinase (Csk), Ret, PI4KIII-α, Skittles and Pkaap were also identified as candidates for further studies. We focused our analysis on Csk and determined Csk is a regulator of Gliotactin endocytosis and plays a role in the regulation of Gliotactin at the TCJ. Although Csk is known as a negative regulator of Src kinases, we identified that the effect of Csk on Gliotactin is independent of Src, and likely occur through an AJ-associated complex. Taken together, this thesis provides novel insights on the function of Csk and identifys other candidate kinases that have the potential to regulate localization and/or signaling events associated with TCJ formation and function.

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The focus of this thesis is to understand the regulatory mechanisms of that control permeability barriers in epithelia. A key role of epithelia is to maintain permeability barriers between tissues. Epithelial junctions are formed to establish a functional barrier between the cells and to ensure cell-cell adhesion. In Drosophila, the tricellular junction (TCJ) generates a barrier at the contact site of three adjacent epithelial cells. Gliotactin is localized exclusively at the corner of three epithelial cells and loss of Gliotactin from the TCJ disrupts the barrier function. Conversely, overexpression of Gliotactin triggers the spread of Gliotactin away from the TCJ leading to apoptosis, delamination, overproliferation and cell migration. Therefore, the expression level of Gliotactin needs to be tightly regulated. Gliotactin protein levels are controlled by tyrosine phosphorylation and subsequent protein endocytosis and degradation. Here we found that Gliotactin expression is also tightly regulated at the mRNA level through microRNA-184. miR-184 targets the Gliotactin 3’UTR and other septate junction mRNAs including NrxIV and Mcr. Gliotactin overexpression triggers BMP signaling through inhibition of Dad, an inhibitory SMAD, and activation of the Tkv type-I receptor and Mad. Elevated level of phosphorylated MAD leads to induction of miR-184 expression. Regulation of Gliotactin at the TCJ is mediated through a Gliotactin-BMP-miR184 feedback loop. We identified a new complex at the TCJ, which regulates junction assembly and function. The scaffolding proteins Scribbled (Scrib) and Discs Large (Dlg) are in close proximity with two TCJ components, Gliotactin and Bark beetle (Bark). The presence of the Scrib PDZ1-2 and the Dlg GUK domains are required for proper formation of the TCJ complex. Loss of Bark or Gliotactin from the TCJ leads to basolateral spread of Scrib and Dlg, while Scrib or Dlg knockdown disrupts the integrity of the complex and promotes the loss of Bark or Gliotactin from the TCJ. Our proposed model suggests that Scrib and Dlg recruit Bark to the TCJ, which in turn leads to Gliotactin recruitment to the TCJ. Overall, we propose that tricellular junction is regulated through two distinct mechanisms, signaling and scaffolding.

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The nervous system is surrounded by neural lamella composed of large glycoproteins including perlecan, collagen and laminin, which bind to underlying perineurial glial cells. The function of perineurial glia and their interaction with the neural lamella is just beginning to be elucidated. Previous studies have demonstrated that integrin is critical for glial wrapping in both vertebrates and Drosophila. Therefore, we have focused on perineurial glia and the role of laminin, an integrin ligand, and basigin, a transmembrane protein known to interact with integrin. Laminin is a heterotrimer composed of an alpha (LanA or Wb), beta (LanB1) and gamma (LanB2) subunit and we demonstrate that LanB2 loss in glia results in accumulation of LanB1 leading to distended ER, ER stress and glial swelling in addition to decreased larval locomotion and lethality. Loss of LanB2 in wrapping glia affected glial ensheathment of axons but surprisingly not larval locomotion. We found that Tango1, a protein thought to exclusively mediate collagen secretion, is also important for laminin secretion in glia via a collagen-independent mechanism. We conclude that it is the loss of one laminin subunit that leads to deleterious consequences through the accumulation of the remaining subunits. Basigin is a highly conserved transmembrane protein involved in cancer metastasis, however its developmental roles are just beginning to be elucidated. We show that basigin is specifically expressed in perineurial glia where it is present in a complex with integrin. Basigin knockdown resulted in a shortened ventral nerve cord, ruffles in the peripheral nerves as well as a collapsed actin cytoskeleton and a redistribution of myosin motors in perineurial glia. We examined the domains within basigin that are required for association with integrin and also examined the effect of basigin knockdown on integrin-associated proteins. Together, the results in this thesis highlight the role of perineurial glia in secreting laminin, facilitating larval locomotion and regulating both central and peripheral nervous system morphology in Drosophila. Due to the conserved structure and function of glia between vertebrates and Drosophila, these results will help direct future research on how perineurial glia regulate nervous system development.

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This thesis is the first in-depth investigation into the roles of Na/K ATPase in assembly and maintenance of septate junctions (SJ) and tricellular junctions (TCJ). Together, these domains block the flow of fluids or pathogens across an epithelia. Loss of either domain (SJ or TCJ) leads to a loss of the permeability barriers. These domains contain large protein complexes that include both the alpha and beta subunits of Na/K ATPase of which the alpha subunit has the potential for scaffolding protein complexes and cell signaling. In cultured fibroblasts the alpha subunit binds and inhibits Src kinase and downstream signaling pathways, but the role in developing epithelia is unknown. Likewise how the Na/K ATPase interacts with other junctional components to establish, maintain and regulate SJs and TCJs is unknown. We examined the consequences of loss of Na/K ATPase in the simple epithelium of the Drosophila wing imaginal disc to understand the role of the pump in mediating cell signaling and scaffolding of SJ and TCJ components. Using RNAi-mediated knockdown of either Na/K ATPase subunit, we observed an increase in activation of the tyrosine kinases, Src and Abl, leading to JNK-mediated apoptosis and apoptosis-induced proliferation. The role of Na/K ATPase in regulating Src activation is conserved between vertebrates and invertebrates and functions to regulate epithelia in vivo. An additional finding was that RNAi knockdown of Na/K ATPase in the imaginal wing disc led to changes in SJ and TCJ proteins that were distinct from knockdown of another core protein, NeurexinIV. In particular the Na/K ATPase led to a specific increase of the TCJ proteins Discs-large and Gliotactin at the TCJ. Overexpression of Gliotactin leads to JNK-mediated apoptosis and cell spreading and these were suppressed by loss of Na/K ATPase but enhanced by loss of NrxIV. This suggests Na/K ATPase has a unique role in both regulation and maintenance of the TCJ. Overall our data support a role for Na/K ATPase both as a scaffolding protein that organizes a subset of SJ and TCJ proteins and as a signaling component in epithelial cell survival.

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Glia are well known for providing essential physical and metabolic support to neurons, as well as regulating neuronal development. Glial development is also modulated by external signals from other cells and the extracellular matrix (ECM). Many signals are transduced into glia by specific receptors, such as integrins for the ECM. Previous studies show that integrins are expressed by all major vertebrate glial subtypes and play key roles in many important developmental processes. However complex composition of the integrin family and difficulties of manipulating genes in vertebrates limit the understanding of in vivo functions of integrins in glia.Drosophila melanogaster is an excellent model for genetic analysis of the nervous system development. In this dissertation, I investigated integrin function in Drosophila glia. Integrins are expressed by glia in both the central and peripheral nervous systems at larval stages, where they form complexes with Talin and Integrin-Linked-Kinase (ILK). I found that integrin complexes were localized to different glia layers in the larval peripheral nerve and optic stalk. By using MARCM and RNA interference techniques, I found that integrins are required for multiple developmental events in individual and populated glia. In the peripheral nerve, integrins are important for glial ensheathment. When integrins were removed, perineurial glia failed to initiate or maintain their wrapping around the nerve and wrapping glia failed to send out numerous membrane processes between axons. In the optic stalk, integrins were necessary for glial migration, deposition and barrier formation. Removal of integrins impaired glial migration into the eye disc. Moreover, perineurial glia tended to aggregate at the anterior half and form multiple layers, and carpet glia failed to form organized septate junctions along the optic stalk. These glial defects resulted in photoreceptor axonal stalling in the eye disc and optic stalk, and mis-targeting in the brain. My work suggests that integrins are important for different aspects of Drosophila glial development and reveals a new glial function in helping photoreceptor axons through the optic stalk. Integrin distribution implicated that integrins may mediate glia-glia or glia-neuron interactions through ECM and non-ECM ligands.

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The focus of this thesis is to investigate the integration of cell division with the septate junction domain in the Drosophila imaginal wing disc epithelium. Columnar epithelia of the imaginal wing disc exhibit complex architecture due to an elaborate series of junctions that are found throughout the membrane. During cell division, these junctions are maintained while new junctions are established; however, their role and influence during mitosis is unclear. This thesis shows that the septate junctions are essential for cytokinesis and Gliotactin at the tricellular junctions is necessary to localize cell division to the septate junction domain, and illustrates a unique role for Gliotactin and the septate junctions outside their classic role of maintaining a permeability barrier. The septate junctions are basolaterally localized transmembrane junctions required in epithelial cells to form a permeability barrier. Although the septate junctions are formed by a large protein complex, this thesis only investigates the three core SJ proteins, NeurexinIV (NrxIV), Coracle (Cor), and Neuroglian (Nrg). Gliotactin (Gli), a Drosophila Neuroligin homologue, is a septate junction associated protein concentrated at the tricellular junction (TCJ), which is necessary to maintain the septate junction permeability barrier. Loss of any of the septate junction proteins, or Gliotactin, leads to structural disruption of the septate junctions and loss of the permeability barrier in a wide range of epithelial derived tissues. Chapter two examines the process of cell division in epithelial cells of the wing imaginal disc with respect to the septate junctions and tricellular junction. Chapter three looks at the role of Gliotactin in maintaining the plane of cell division within the septate junction domain, and chapter four shows that the septate junctions are necessary for ingression furrow stability and the association of the contractile ring with the membrane during late cytokinesis. This work demonstrates a novel role for the septate and tricellular junctions during mitosis in Drosophila, which has implications for the role of tight junctions in vertebrate cells.

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Corticofugal axons projecting to the thalamus, brainstem and spinal cord must travel the same initial trajectory through the subcortical lateral and medial ganglionic eminences, and are therefore likely subject to the same sets of guidance cues. These cues direct the course of corticofugal axons bringing them to each intermediate target, until they reach the diencephalic-telencephalic boundary, where axons targeting the thalamus turn dorsally and brainstem and spinal cord targeting axons turn ventrally. Many of these guidance cues have been elucidated, yet there are still gaps in our understanding of the formation of the corticofugal projection. I found that Sema5B expression flanked the presumptive internal capsule during its formation, and was therefore ideally situated both spatially and temporally to act as an instructive cue for descending cortical axons. In Chapter 2, I show that Sema5B is not only capable of inhibiting cortical axons in vitro, but can cause misguidance of cortical axons in slice culture when placed ectopically over normally non-Sema5B expressing regions. In addition, I show that the loss of Sema5B from the neocortical VZ resulted in aberrant penetration of this normally avoided region. Therefore Sema5B is both necessary and sufficient to inhibit the corticofugal projection. Semaphorins and their plexin receptors are frequently proteolyzed to modulate the elicited responses in navigating growth cones. In Chapter 3 I show that Sema5B cleavage results in an inhibitory fragment that in heterologous cells can produce inhibitory gradients for cortical explants in collagen gel co-cultures, and collapse dissociated cortical neuronal growth cones, an effect that can be blocked with a function disrupting antibody to the cell adhesion molecule TAG-1. This thesis shows that Sema5B, a guidance cue with a hitherto unknown function, is responsible for a very important aspect of cortical development. My work leads to a final proposal that Sema5B is in fact a two-in-one protein with separable inhibitory and alternate complex functions, the implications of which are discussed thoroughly in Chapter 4.

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