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是如何包裹和保护周围神经的。我们研究了果蝇神经胶质细胞发育的机制，其中每条周围神经都由几层神经胶质细胞隔绝，而隔绝对神经系统功能是必不可少的。人们对触发神经胶质细胞沿轴突迁移并包裹轴突或包围和支持神经的机制知之甚少。胶质包被的形成涉及神经胶质细胞、神经胶质轴突和神经胶质细胞外基质(ECM)之间的黏附和沟通，并伴随着细胞骨架的广泛重排。在果蝇中，成熟的周围神经有三个不同的胶质层，神经胶质鞘必须保持和扩张，以适应动物的生长。由于血脑屏障(BBB)的丧失，神经胶质细胞黏附的丧失破坏了神经胶质鞘，导致瘫痪和死亡，但鞘的形成和维持还不是很清楚。胶质细胞-细胞外基质相互作用的丧失会导致中枢神经系统结构和周围神经完整性的破坏。我研究的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。*我们由NSERC资助的研究发现了对建立神经胶质鞘至关重要的蛋白质复合体。我们发现整合素是周围神经系统中胶质细胞-胶质细胞和胶质细胞-细胞外基质通讯的关键，并维持神经胶质鞘和血脑屏障。我们发现basigin(BSG)调节整合素介导的胶质细胞与ECM的黏附。我们确定钙粘附素确保神经胶质包被和血脑屏障的形成。有趣的是，钙粘附素和整合素在这一过程中共同发挥作用，了解这些复合体如何控制神经胶质包膜是我们研究计划的下一步。目前拨款的两个短期目标是确定以下机制：*目的1)在鞘和血脑屏障形成过程中整合素和钙粘素功能的收敛。*目的2)连接整合素和basigin以调节神经胶质细胞-细胞外基质的相互作用，并确保周围神经的结构完整性。*我的实验室在研究外周神经胶质细胞发育和鞘形成方面有丰富的经验。我们已经生成了一套遗传工具，允许我们操纵三个外周胶质细胞层中每一个表达的基因，然后评估对神经系统功能和形态的结果。我们利用多方面的方法，结合遗传学(包括RNAi、CRISPR基因组编辑)、细胞生物学(包括荧光团标记的内源蛋白质、基于FRET的张力传感器)、生物化学(包括邻近连接分析)、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.**