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In JoVE (1)
Other Publications (12)
- Development (Cambridge, England)
- The Journal of Cell Biology
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Proteins
- Developmental Biology
- Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
- Frontiers in Bioscience : a Journal and Virtual Library
- Seminars in Cell & Developmental Biology
- Journal of Cell Science
- Neuron Glia Biology
- Journal of Cell Science
- Development (Cambridge, England)
Articles by Vanessa J. Auld in JoVE
JoVE General
Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc
Department of Zoology, University of British Columbia - UBC
Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.
Other articles by Vanessa J. Auld on PubMed
RhoA and Rac1 GTPases Mediate the Dynamic Rearrangement of Actin in Peripheral Glia
Peripheral glial cells in both vertebrates and insects are born centrally and travel large distances to ensheathe axons in the periphery. There is very little known about how this migration is carried out. In other cells, it is known that rearrangement of the Actin cytoskeleton is an integral part of cell motility, yet the distribution of Actin in peripheral glial cell migration in vivo has not been previously characterized. To gain an understanding of how glia migrate, we specifically labeled the peripheral glia of Drosophila melanogaster using an Actin-GFP marker and analyzed their development in the embryonic PNS. It was found that Actin cytoskeleton is dynamically rearranged during glial cell migration. The peripheral glia were observed to migrate as a continuous chain of cells, with the leading glial cells appearing to participate to the greatest extent in exploring the extracellular surroundings with filopodia-like Actin containing projections. We hypothesized that the small GTPases Rho, Rac and Cdc42 are involved in Actin cytoskeletal rearrangements that underlie peripheral glial migration and nerve ensheathement. To test this, transgenic forms of the GTPases were ectopically expressed specifically in the peripheral glia during their migration and wrapping phases. The effects on glial Actin-GFP distribution and the overall effects on glial cell migration and morphological development were assessed. We found that RhoA and Rac1 have distinct roles in peripheral glial cell migration and nerve ensheathement; however, Cdc42 does not have a significant role in peripheral glial development. RhoA and Rac1 gain-of-function and loss-of-function mutants had both disruption of glial cell development and secondary effects on sensory axon fasciculation. Together, Actin cytoskeletal dynamics is an integral part of peripheral glial migration and nerve ensheathement, and is mediated by RhoA and Rac1.
Gliotactin, a Novel Marker of Tricellular Junctions, is Necessary for Septate Junction Development in Drosophila
Septate junctions (SJs), similar to tight junctions, function as transepithelial permeability barriers. Gliotactin (Gli) is a cholinesterase-like molecule that is necessary for blood-nerve barrier integrity, and may, therefore, contribute to SJ development or function. To address this hypothesis, we analyzed Gli expression and the Gli mutant phenotype in Drosophila epithelia. In Gli mutants, localization of SJ markers neurexin-IV, discs large, and coracle are disrupted. Furthermore, SJ barrier function is lost as determined by dye permeability assays. These data suggest that Gli is necessary for SJ formation. Surprisingly, Gli distribution only colocalizes with other SJ markers at tricellular junctions, suggesting that Gli has a unique function in SJ development. Ultrastructural analysis of Gli mutants supports this notion. In contrast to other SJ mutants in which septa are missing, septa are present in Gli mutants, but the junction has an immature morphology. We propose a model, whereby Gli acts at tricellular junctions to bind, anchor, or compact SJ strands apically during SJ development.
Reciprocal Interactions Between Neurons and Glia Are Required for Drosophila Peripheral Nervous System Development
A major developmental role of peripheral glia is to mediate sensory axon guidance; however, it is not known whether sensory neurons influence peripheral glial development. To determine whether glia and neurons reciprocally interact during embryonic development, we ablated each cell type by overexpressing the apoptosis gene, grim, and observed the effects on peripheral nervous system (PNS) development. When neurons are ablated, glial defects occur as a secondary effect, and vice versa. Therefore glia and neurons are codependent during embryogenesis. To further explore glial-neuronal interactions, we genetically disrupted glial migration or differentiation and observed the secondary effects on sensory neuron development. Glial migration and ensheathment of PNS axons was blocked by overexpression of activated Rho GTPase, a regulator of actin dynamics. Here, sensory axons extended to the CNS without exhibiting gross pathfinding errors. In contrast, disrupting differentiation by expression of dominant-negative Ras GTPase in glia resulted in major sensory axon pathfinding errors, similar to those seen in glial ablations. Glial overexpression of transgenic components of the epidermal growth factor receptor (EGFR) signaling pathway yielded similar sensory neuron defects and also downregulated the expression of the glial marker Neuroglian. Mutant analysis also suggested that the EGFR ligands Spitz and Vein play roles in peripheral glial development. The observations support a model in which glia express genes necessary for sensory neuron development, and these genes are potentially under the control of the EGFR/Ras signaling pathway.
The Intracellular Domain of the Drosophila Cholinesterase-like Neural Adhesion Protein, Gliotactin, is Natively Unfolded
Drosophila gliotactin (Gli) is a 109-kDa transmembrane, cholinesterase-like adhesion molecule (CLAM), expressed in peripheral glia, that is crucial for formation of the blood-nerve barrier. The intracellular portion (Gli-cyt) was cloned and expressed in the cytosolic fraction of Escherichia coli BLR(DE3) at 45 mg/L and purified by Ni-NTA (nitrilotriacetic acid) chromatography. Although migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), under denaturing conditions, was unusually slow, molecular weight determination by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) confirmed that the product was consistent with its theoretical size. Gel filtration chromatography yielded an anomalously large Stokes radius, suggesting a fully unfolded conformation. Circular dichroism (CD) spectroscopy demonstrated that Gli-cyt was >50% unfolded, further suggesting a nonglobular conformation. Finally, 1D-(1)H NMR conclusively demonstrated that Gli-cyt possesses an extended unfolded structure. In addition, Gli-cyt was shown to possess charge and hydrophobic properties characteristic of natively unfolded proteins (i.e., proteins that, when purified, are intrinsically disordered under physiologic conditions in vitro).
Transient Apical Polarization of Gliotactin and Coracle is Required for Parallel Alignment of Wing Hairs in Drosophila
In Drosophila, wing hairs are aligned in a distally oriented, parallel array. The frizzled pathway determines proximal-distal cell polarity in the wing; however, in frizzled pathway mutants, wing hairs remain parallel. How wing hairs align has not been determined. We have demonstrated a novel role for the septate junction proteins Gliotactin (Gli) and Coracle (Cora) in this process. Prior to prehair extension, Gli and Cora were restricted to basolateral membranes. During pupal prehair development, Gli and Cora transiently formed apical ribbons oriented from the distal wing tip to the proximal hinge. These ribbons were aligned beneath prehair bases and persisted for several hours. During this time, Gli was lost entirely from the basolateral domain. A Gliotactin mutation altered the apical polarization Gli and Cora and induced defects in hair alignment in pupal and adult stages. Genetic and cell biological assays demonstrated that Gli and Cora function to align hairs independently of frizzled. Taken together, our results indicate that Gli and Cora function as the first-identified members of a long-predicted, frizzled-independent parallel alignment mechanism. We propose a model whereby the apical polarization of Gli and Cora functions to stabilize and align prehairs relative to anterior-posterior cell boundaries during pupal wing development.
Signaling in Glial Development: Differentiation Migration and Axon Guidance
Glial cells have diverse functions that are necessary for the proper development and function of complex nervous systems. During development, a variety of reciprocal signaling interactions between glia and neurons dictate all parts of nervous system development. Glia may provide attractive, repulsive, or contact-mediated cues to steer neuronal growth cones and ensure that neurons find their appropriate synaptic targets. In fact, both neurons and glia may act as migrational substrates for one another at different times during development. Also, the exchange of trophic signals between glia and neurons is essential for the proper bundling, fasciculation, and ensheathement of axons as well as the differentiation and survival of both cell types. The growing number of links between glial malfunction and human disease has generated great interest in glial biology. Because of its relative simplicity and the many molecular genetic tools available, Drosophila is an excellent model organism for studying glial development. This review will outline the roles of glia and their interactions with neurons in the embryonic nervous system of the fly.
Evolution of Clams (cholinesterase-like Adhesion Molecules): Structure and Function During Development
The protein family known as CLAMS (cholinesterase-like adhesion molecules) forms a novel class of heterophilic cell adhesion proteins. Family members are found through a wide range of metazoans and play a role during the development of multiple tissues. The majority of members of this family are transmembrane proteins with an extracellular domain that is conserved with cholinesterases including acetylcholinesterase. Yet all family members lack one or more of the residues that make up the catalytic triad necessary for enzymatic function. Therefore the conserved cholinesterase-like domain is not necessary for enzymatic function but does appear to play a role in heterophilic binding. CLAMS are expressed in a wide array of tissues and most family members appear to play a role in cell adhesion and junction formation. The development of junctions including septate junctions and synaptic junctions require CLAM family members such as Gliotactin and Neuroligins respectively. Modeling of the cholinesterase-like domain reveals that evolutionary changes to the binding pocket of the cholinesterase domain may produce a range of different ligand binding partners for CLAM family members. In this vein, previous chimera experiments and recent work has identified mutations in CLAM family members that affect the structure of the cholinesterase-like domain. These mutant forms affect protein function during the development of specialized junctions and confirm the role of the cholinesterase domain in mediating heterophilic binding.
Roles of Glia in the Drosophila Nervous System
Glial cells have diverse functions that are necessary for the proper development and function of complex nervous systems. Various insects, primarily the fruit fly Drosophila melanogaster and the moth Manduca sexta, have provided useful models of glial function during development. The present review will outline evidence of glial contributions to embryonic, visual, olfactory and wing development. We will also outline evidence for non-developmental functions of insect glia including blood-brain-barrier formation, homeostatic functions and potential contributions to synaptic function. Where relevant, we will also point out similarities between the functions of insect glia and their vertebrate counterparts.
Gliotactin and Discs Large Form a Protein Complex at the Tricellular Junction of Polarized Epithelial Cells in Drosophila
The tricellular junction (TCJ) forms at the convergence of pleated septate junctions (SJs) from three adjacent cells in polarized epithelia and is necessary for maintaining the transepithelial barrier. In Drosophila, the transmembrane protein Gliotactin was the first identified marker of the TCJ, but little is known about other molecular constituents. We now show that Gliotactin associates with Discs large at the TCJ in a Ca(2+)-dependent manner. Discs large is essential for the formation of the TCJ and the localization of Gliotactin. Surprisingly, Gliotactin localization at the TCJ was independent of its PDZ-binding motif and Gliotactin did not bind directly to Discs large. Therefore Gliotactin and Discs large association is through intermediary proteins at the TCJ. Gliotactin can associate with other septate junction proteins but this was detected only when Gliotactin was overexpressed and spread throughout the septate junction domain. Gliotactin overexpression and spread also resulted in a reduction of Discs large staining but not vice versa. These results suggest that Discs large participates in different protein interactions in the SJ and the TCJ. Finally this work supports a model where Gliotactin and Dlg are components of a larger protein complex that links the converging SJs with the TCJ to create the transepithelial barrier.
No Pun Intended: Future Directions in Invertebrate Glial Cell Migration Studies
Glial cells play a wide range of essential roles in both nervous system development and function and has been reviewed recently (Parker and Auld, 2006). Glia provide an insulating sheath, either form or direct the formation of the blood-brain barrier, contribute to ion and metabolite homeostasis and provide guidance cues. Glial function often depends on the ability of glial cells to migrate toward specific locations during nervous system development. Work in nervous system development in insects, in particular in the fruit fly Drosophila melanogaster and the tobacco hornworm Manduca sexta, has provided significant insight into the roles of glia, although the molecular mechanisms underlying glial cell migration are being determined only now. Indeed, many of the processes and mechanisms discovered in these simpler systems have direct parallels in the development of vertebrate nervous systems. In this review, we first examine the developmental contexts in which invertebrate glial cell migration has been observed, we next discuss the characterized molecules required for proper glial cell migration, and we finally discuss future goals to be addressed in the study of glial cell development.
Control of Gliotactin Localization and Levels by Tyrosine Phosphorylation and Endocytosis is Necessary for Survival of Polarized Epithelia
The tricellular junction (TCJ) forms at the convergence of bicellular junctions from three adjacent cells in polarized epithelia and is necessary for maintaining the transepithelial barrier. In the fruitfly Drosophila, the TCJ is generated at the meeting point of bicellular septate junctions. Gliotactin was the first identified component of the TCJ and is necessary for TCJ and septate junction development. Gliotactin is a member of the neuroligin family and associates with the PDZ protein discs large. Beyond this interaction, little is known about the mechanisms underlying Gliotactin localization and function at the TCJ. In this study, we show that Gliotactin is phosphorylated at conserved tyrosine residues, a process necessary for endocytosis and targeting to late endosomes and lysosomes for degradation. Regulation of Gliotactin levels through phosphorylation and endocytosis is necessary as overexpression results in displacement of Gliotactin away from the TCJ throughout the septate junction domain. Excessive Gliotactin in polarized epithelia leads to delamination, paired with subsequent migration, and apoptosis. The apoptosis and the resulting compensatory proliferation resulting from high levels of Gliotactin are mediated by the Drosophila JNK pathway. Therefore, Gliotactin levels within the cell membrane are regulated to ensure correct protein localization and cell survival.
Integrins Are Necessary for the Development and Maintenance of the Glial Layers in the Drosophila Peripheral Nerve
Peripheral nerve development involves multiple classes of glia that cooperate to form overlapping glial layers paired with the deposition of a surrounding extracellular matrix (ECM). The formation of this tubular structure protects the ensheathed axons from physical and pathogenic damage and from changes in the ionic environment. Integrins, a major family of ECM receptors, play a number of roles in the development of myelinating Schwann cells, one class of glia ensheathing the peripheral nerves of vertebrates. However, the identity and the role of the integrin complexes utilized by the other classes of peripheral nerve glia have not been determined in any animal. Here, we show that, in the peripheral nerves of Drosophila melanogaster, two integrin complexes (αPS2βPS and αPS3βPS) are expressed in the different glial layers and form adhesion complexes with integrin-linked kinase and Talin. Knockdown of the common beta subunit (βPS) using inducible RNAi in all glial cells results in lethality and glial defects. Analysis of integrin complex function in specific glial layers showed that loss of βPS in the outermost layer (the perineurial glia) results in a failure to wrap the nerve, a phenotype similar to that of Matrix metalloproteinase 2-mediated degradation of the ECM. Knockdown of βPS integrin in the innermost wrapping glia causes a loss of glial processes around axons. Together, our data suggest that integrins are employed in different glial layers to mediate the development and maintenance of the protective glial sheath in Drosophila peripheral nerves.
