Genetic dissection of peripheral glia and glial sheath development

我的研究计划的长期目标是了解胶质细胞如何包膜和保护周围神经。我们研究了黑腹果蝇神经胶质发育的机制，其中每个周围神经都被神经胶质层隔离，隔离对神经系统功能至关重要。关于胶质细胞沿轴突或包围并支持神经迁移的机制，我们知之甚少。胶质膜的形成涉及胶质-胶质细胞、胶质-轴突和胶质-细胞外基质（ECM）之间的粘附和通讯，并伴有细胞骨架的广泛重排。在果蝇中，三个不同的胶质层有助于成熟的周围神经，胶质鞘必须保持和扩张以匹配动物的生长。由于血脑屏障（BBB）的丧失，神经胶质瘤-神经胶质瘤黏附的丧失会破坏神经胶质瘤鞘，导致瘫痪和死亡，然而，神经胶质瘤鞘是如何形成和维持的还不清楚。神经胶质- ecm相互作用的丧失导致中枢神经系统结构和周围神经完整性的破坏。我的研究目的是确定神经胶质鞘形成的机制，并测试这些高度保守的过程的破坏对神经系统功能的功能后果。我们NSERC资助的研究确定了对建立神经胶质鞘至关重要的蛋白质复合物。我们发现整合素是外周神经系统中胶质-胶质细胞和胶质- ecm通讯的关键，并维持胶质鞘和血脑屏障。我们发现Basigin （Bsg）调节整合素介导的胶质细胞粘附到ECM。我们确定钙粘蛋白确保了胶质膜和血脑屏障的形成。有趣的是，钙粘蛋白和整合蛋白在这个过程中一起起作用，了解这些复合物如何控制胶质鞘是我们研究计划的下一步。当前拨款的两个短期目标是确定以下机制：目标1)整合素和钙粘蛋白在鞘和血脑屏障形成中的功能趋同。目的2)连接整合素和Basigin调节胶质细胞与ecm的相互作用，确保周围神经的结构完整性。我的实验室对外周胶质细胞的发育和鞘的形成有着丰富的研究经验。我们已经生成了一套基因工具，使我们能够操纵在三个周围神经胶质层中表达的基因，然后评估神经系统功能和形态的结果。我们利用多方面的方法，包括遗传学（包括RNAi， CRISPR基因组编辑），细胞生物学（包括荧光团标记的内源性蛋白质，基于FRET的张力传感器），生物化学（包括近距离连接测定），光遗传学来调节轴突活动，超分辨率成像和行为研究。因此，我们使用果蝇模型来描述所有动物神经胶质细胞发育和鞘形成的基本机制。

The long-term goal of my research program is to understand how glia ensheath and protect the peripheral nerve. We study the mechanisms that underlie glia development in Drosophila melanogaster, where each peripheral nerve is insulated by layers of glia and insulation is essential for nervous system function. Little is known about the mechanisms that trigger glia to migrate along and wrap axons or surround and support the nerve. The formation of the glial wrap involves adhesion and communication between glia-glia, glia-axon and glia-extracellular matrix (ECM) paired with extensive rearrangements of the cytoskeleton. In Drosophila three distinct glial layers contribute to the mature peripheral nerve and the glial sheath must be maintained and expand to match animal growth. Loss of glia-glia adhesion disrupts the glial sheath leading to paralysis and lethality due to loss of the blood-brain barrier (BBB), yet how the sheath forms and is maintained is not well understood. Loss of the glia-ECM interaction leads to disruption of the structure of the CNS and peripheral nerve integrity. The aim of my research is to identify the mechanisms underlying glial sheath formation and test the functional consequences that disruption of these highly conserved processes have on nervous system function. Our NSERC funded research identified protein complexes critical to establish the glial sheath. We found that integrins are key to glia-glia and glia-ECM communication in the peripheral nervous system and maintain the glial sheath and the BBB. We found Basigin (Bsg) regulates integrin-mediated glial adhesion to the ECM. We determined that Cadherins ensure the formation of the glial wrap and BBB. Of interest, Cadherins and Integrins function together in this process and understanding how these complexes control glial ensheathment is the next step in our research program. The two short term goals of the current grant are to determine the mechanisms that: Aim 1) Underly the convergence of integrin and cadherin function in sheath and BBB formation. Aim 2) Link integrins and Basigin to modulate glia-ECM interactions and ensure the structural integrity of the peripheral nerve. My laboratory has extensive experience studying peripheral glia development and sheath formation. We have generated a suite of genetic tools that allow us to manipulate the genes expressed in each of the three peripheral glia layers and then assess the results on nervous system function and morphology. We utilize a multi-faceted approach that incorporates genetics (including RNAi, CRISPR genome editing), cell biology (including fluorophore-tagged endogenous proteins, FRET based tension sensors), biochemistry (including proximity ligation assays), optogenetics to regulate axonal activity, super-resolution imaging and behaviourial studies.  Thus we use the Drosophila model to characterize the fundamental mechanisms that underlie glial cell development and sheath formation in all animals.