果蝇黑色素血脑屏障完整性分析
Summary
血脑屏障完整性对神经系统功能至关重要。在果蝇黑色素仪中，血脑屏障是由胚胎发育晚期的胶质细胞形成的。该协议描述了在D.melanogaster胚胎和第三星幼虫中进行血脑屏障形成和维护的测定方法。
Abstract
适当的神经系统发展包括形成血脑屏障，扩散屏障，严格调节进入神经系统，保护神经组织免受毒素和病原体的侵害。这种屏障形成的缺陷与神经病变有关，在许多神经退行性疾病中观察到这种屏障的分解。因此，确定调节血脑屏障形成和维护的基因以确定潜在的治疗目标至关重要。为了了解这些基因在神经发育中的确切作用，有必要分析改变的基因表达对血脑屏障完整性的影响。许多在建立血脑屏障中发挥作用的分子被发现被保存在真核物种，包括果蝇，果蝇，果蝇黑色素。果蝇已被证明是一个优秀的模型系统，用于检查调节神经系统发育和功能的分子机制。该协议描述了在D.黑色素气症发育的胚胎和幼虫阶段，用于血液脑屏障完整性的分步测定程序。
Introduction
在发育过程中，细胞-细胞的通信和相互作用对组织和器官结构和功能的建立至关重要。在某些情况下，这些细胞-细胞相互作用密封器官从周围环境，以确保适当的器官功能。神经系统就是这种情况，神经系统被血脑屏障（BBB）绝缘。人体BBB功能障碍与包括癫痫在内的神经系统疾病有关，在神经退行性疾病（包括多发性硬化症和肌萎缩性侧索硬化症1）中观察到屏障的分解。在哺乳动物中，BBB是由内皮细胞2、3之间的紧密结形成。其他动物，包括果蝇，果蝇黑色素，有一个由胶质细胞组成的BBB。这些胶质细胞形成选择性渗透屏障，控制营养物质、废物、毒素和大分子进入和流出神经系统的运动4。这允许保持发射作用电位所需的电化学梯度，允许移动和协调4。在D.melanogaster中，胶质保护神经系统免受富含钾的、血样淋巴5的淋巴。
在D.melanogaster的中枢神经系统（CNS）和周围神经系统（PNS）中，两个外层胶质层、下眼胶质和眼质胶质，以及细胞外基质的外网络，神经层状，形成淋巴脑和淋巴神经屏障6，在本文中称为BBB。在发育过程中，亚倍体成为多倍体并扩大以包围神经系统5，6，7，8，9，10，11.亚苯并流感形成分离结，提供淋巴和神经系统5、6、12之间的主要扩散屏障。这些结在分子上类似于脊椎动物在骨髓胶质的副节点处发现的类似分离的结，它们的作用与哺乳动物13、14的BBB中的紧密结相同。15,16,17.环状胶质分裂、生长和包裹在亚佩里尼胶质周围，以调节代谢物和大分子6、10、18、19的扩散。BBB形成在25°C5，8下产卵（AEL）后18.5小时完成。先前的研究已经确定了BBB形成的关键调控基因20，21，22。为了更好地了解这些基因的确切作用，必须研究这些潜在调控器突变对BBB完整性的影响。虽然以前的研究已经概述了在胚胎和幼虫中测定BBB完整性的方法，但是这个测定的综合方案还没有被描述5，7。此分步协议描述了在D.黑色素气芽期和第三星幼虫阶段对BBB完整性进行诊断的方法。
Protocol
1. 样品收集 胚胎收集 在每个胚胎收集笼中，使用至少50名处女和20-25名男性进行采集。在开始收集23之前，用玉米粉-琼脂食品（材料表）在瓶子里孵育这些苍蝇1⁄2天。注：可以使用更多的苍蝇，但雌性与雄性的比例应保持在2：1。 预加热苹果汁琼脂板 （表 1） 在 25 °C 过夜.注：这是适?…
Representative Results
此处描述的方法允许在D.黑色素气素胚胎和幼虫中在整个CNS中实现BBB完整性的可视化（图1）。在胚胎发育晚期完成BBB形成后，BBB功能从大脑中排除大分子和VNC5。该协议利用此功能来测定BBB的形成。当野生型（俄勒冈R）后期17（20⁄21 h老）胚胎被注射10kDa dextran结合成磺胺101氯化氯化荧光染料时，大dextran分子被排除在VNC中，如预期（图2…
Discussion
该协议全面描述了在D.melanogaster发育的晚期和第三星幼虫阶段，分析BBB完整性所需的步骤。其他地方也曾描述过类似的方法，以在发展期间以及成人阶段5、7、29、30中，分析BBB的完整性。但是，材料和方法部分中对程序的描述通常很广泛，缺乏足够的细节，便于实施，因此需要代表研究人员进?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢F·布赖恩·皮克特博士和罗德尼·戴尔博士使用注射设备。这项工作由芝加哥洛约拉大学向法学博士学位、D.T.和J.J.的研究资助。
Materials
| 10 kDa sulforhodamine 101 acid chloride (Texas Red) Dextran | ThermoFisher Scientific | D1863 | Dextran should be diluted in autoclaved ddH2O to a concentration of 25 mg/mL. |
| 20 μL Gel-Loading Pipette Tips | Eppendorf | 22351656 | |
| 100% Ethanol (200 proof) | Pharmco-Aaper | 11000200 | |
| Active Dry Yeast | Red Star | ||
| Agar | Fisher Scientific | BP1423 | |
| Agarose | Fisher Scientific | BP160-500 | |
| Air Compressor | DeWalt | D55140 | |
| Apple Juice | Mott's Natural Apple Juice | ||
| Bleach | Household Bleach | 1-5% Hypochlorite | |
| Borosilicate Glass Capillaries | World Precision Instruments | 1B100F-4 | |
| Bottle Plugs | Fisher Scientific | AS-277 | |
| Cell Strainers | BD Falcon | 352350 | |
| Confocal Microscope | Olympus | FV1000 | Samples imaged using 20x objective (UPlanSApo 20x/ 0.75) |
| Cotton-Tipped Applicator | Puritan | 19-062614 | |
| Double-Sided Tape 1/2" | Scotch | ||
| Dumont Tweezers; Pattern #5; .05 X .01mm Tip | Roboz | RS-5015 | |
| Fly Food Bottles | Fisher Scientific | AS-355 | |
| Fly Food Vials | Fisher Scientific | AS-515 | |
| Foot Pedal | Treadlite II | T-91-S | |
| Gel Caster | Bio-Rad | 1704422 | |
| Gel Tray | Bio-Rad | 1704436 | |
| Glass Pipette | VWR | 14673-010 | |
| Glycerol | Fisher Scientific | BP229-1 | |
| Granulated sugar | Purchased from grocery store. | ||
| Halocarbon Oil | Lab Scientific, Inc. | FLY-7000 | |
| Light Source | Schott | Ace I | |
| Manipulator Stand | World Precision Instruments | M10 | |
| Micromanipulator | World Precision Instruments | KITE-R | |
| Micropipette Puller | Sutter Instrument Co. | P-97 | |
| Needle Holder | World Precision Instruments | MPH310 | |
| Nightsea Filter Sets | Electron Microscopy Science | SFA-LFS-CY | For visualization of YFP |
| Nightsea Full Adapter System w/ Royal Blue Color Light Head | Electron Microscopy Science | SFA-RB | For visualization of GFP |
| Paintbrush | Simply Simmons | Chisel Blender #6 | |
| Pipetter | Fisher Scientific | 13-683C | |
| Pneumatic Pump | World Precision Instruments | PV830 | This is also referred to as a microinjector or pressure regulator. Since the model used in our study is no longer available this is one alternative. |
| Potassium Chloride | Fisher Scientific | BP366-500 | |
| Potassium Phosphate Dibasic | Fisher Scientific | BP363-500 | |
| Small Embryo Collection Cages | Genesee Scientific | 59-100 | |
| Sodium Chloride | Fisher Scientific | BP358-212 | |
| Sodium Phosphate Dibasic Anhydrous | Fisher Scientific | BP332-500 | |
| Steel Base Plate | World Precision Instruments | 5052 | |
| Stereomicroscope | Carl Zeiss | Stemi 2000 | Used for tissue dissection. |
| Stereomicroscope with transmitted light source | Baytronix | Used for injection. | |
| Tegosept (p-hydroxybenzoic acid, methyl ester) | Genesee Scientific | 20-258 | |
| Triton X-100 | Fisher Scientific | BP151-500 | Nonionic surfactant |
| Vial Plugs | Fisher Scientific | AS-273 |
References
- Obermeier, B., Daneman, R., Ransohoff, R. M. Development, maintenance and disruption of the blood-brain barrier. Nature Medicine. 19 (12), 1584-1596 (2013).
- Brightman, M. W., Reese, T. S. Junctions between intimately apposed cell membranes in the vertebrate brain. Journal of Cell Biology. 40 (3), 648-677 (1969).
- Tietz, S., Engelhardt, B. Brain barriers: Crosstalk between complex tight junctions and adherens junctions. Journal of Cell Biology. 209 (4), 493-506 (2015).
- Hindle, S. J., Bainton, R. J. Barrier mechanisms in the Drosophila blood-brain barrier. Frontiers in Neuroscience. 8, 414 (2014).
- Schwabe, T., Bainton, R. J., Fetter, R. D., Heberlein, U., Gaul, U. GPCR signaling is required for blood-brain barrier formation in drosophila. Cell. 123 (1), 133-144 (2005).
- Stork, T., et al. Organization and function of the blood-brain barrier in Drosophila. Journal of Neuroscience. 28 (3), 587-597 (2008).
- Unhavaithaya, Y., Orr-Weaver, T. L. Polyploidization of glia in neural development links tissue growth to blood-brain barrier integrity. Genes & Development. 26 (1), 31-36 (2012).
- Schwabe, T., Li, X., Gaul, U. Dynamic analysis of the mesenchymal-epithelial transition of blood-brain barrier forming glia in Drosophila. Biology Open. 6 (2), 232-243 (2017).
- Von Stetina, J. R., Frawley, L. E., Unhavaithaya, Y., Orr-Weaver, T. L. Variant cell cycles regulated by Notch signaling control cell size and ensure a functional blood-brain barrier. Development. 145 (3), dev157115 (2018).
- von Hilchen, C. M., Beckervordersandforth, R. M., Rickert, C., Technau, G. M., Altenhein, B. Identity, origin, and migration of peripheral glial cells in the Drosophila embryo. Mechanisms of Development. 125 (3-4), 337-352 (2008).
- Beckervordersandforth, R. M., Rickert, C., Altenhein, B., Technau, G. M. Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as related to lineage and gene expression. Mechanisms of Development. 125 (5-6), 542-557 (2008).
- Bellen, H. J., Lu, Y., Beckstead, R., Bhat, M. A. Neurexin IV, caspr and paranodin–novel members of the neurexin family: encounters of axons and glia. Trends in Neurosciences. 21 (10), 444-449 (1998).
- Baumgartner, S., et al. A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function. Cell. 87 (6), 1059-1068 (1996).
- Banerjee, S., Pillai, A. M., Paik, R., Li, J., Bhat, M. A. Axonal ensheathment and septate junction formation in the peripheral nervous system of Drosophila. Journal of Neuroscience. 26 (12), 3319-3329 (2006).
- Bhat, M. A., et al. Axon-glia interactions and the domain organization of myelinated axons requires neurexin IV/Caspr/Paranodin. Neuron. 30 (2), 369-383 (2001).
- Faivre-Sarrailh, C., et al. Drosophila contactin, a homolog of vertebrate contactin, is required for septate junction organization and paracellular barrier function. Development. 131 (20), 4931-4942 (2004).
- Salzer, J. L., Brophy, P. J., Peles, E. Molecular domains of myelinated axons in the peripheral nervous system. Glia. 56 (14), 1532-1540 (2008).
- von Hilchen, C. M., Bustos, A. E., Giangrande, A., Technau, G. M., Altenhein, B. Predetermined embryonic glial cells form the distinct glial sheaths of the Drosophila peripheral nervous system. Development. 140 (17), 3657-3668 (2013).
- Matzat, T., et al. Axonal wrapping in the Drosophila PNS is controlled by glia-derived neuregulin homolog Vein. Development. 142 (7), 1336-1345 (2015).
- Limmer, S., Weiler, A., Volkenhoff, A., Babatz, F., Klambt, C. The Drosophila blood-brain barrier: development and function of a glial endothelium. Frontiers in Neuroscience. 8, 365 (2014).
- Ho, T. Y., et al. Expressional Profiling of Carpet Glia in the Developing Drosophila Eye Reveals Its Molecular Signature of Morphology Regulators. Frontiers in Neuroscience. 13, 244 (2019).
- DeSalvo, M. K., et al. The Drosophila surface glia transcriptome: evolutionary conserved blood-brain barrier processes. Frontiers in Neuroscience. 8, 346 (2014).
- . BDSC Cornmeal Food Available from: https://bdsc.indiana.edu/information/recipes/bloomfood.html (2017)
- Le, T., et al. A new family of Drosophila balancer chromosomes with a w- dfd-GMR yellow fluorescent protein marker. 유전학. 174 (4), 2255-2257 (2006).
- Casso, D., Ramirez-Weber, F. A., Kornberg, T. B. GFP-tagged balancer chromosomes for Drosophila melanogaster. Mechanisms of Development. 88 (2), 229-232 (1999).
- Halfon, M. S., et al. New fluorescent protein reporters for use with the Drosophila Gal4 expression system and for vital detection of balancer chromosomes. Genesis. 34 (1-2), 135-138 (2002).
- Miller, D. F., Holtzman, S. L., Kaufman, T. C. Customized microinjection glass capillary needles for P-element transformations in Drosophila melanogaster. BioTechniques. 33 (2), 366-372 (2002).
- Luong, D., Perez, L., Jemc, J. C. Identification of raw as a regulator of glial development. PLoS One. 13 (5), e0198161 (2018).
- Pinsonneault, R. L., Mayer, N., Mayer, F., Tegegn, N., Bainton, R. J. Novel models for studying the blood-brain and blood-eye barriers in Drosophila. Methods in Molecular Biology. 686, 357-369 (2011).
- Love, C. R., Dauwalder, B., Barichello, T. Drosophila as a Model to Study the Blood-Brain Barrier. Blood-Brain Barrier. , 175-185 (2019).
- Lin, D. M., Goodman, C. S. Ectopic and increased expression of Fasciclin II alters motoneuron growth cone guidance. Neuron. 13 (3), 507-523 (1994).
- Sepp, K. J., Schulte, J., Auld, V. J. Peripheral glia direct axon guidance across the CNS/PNS transition zone. 발생학. 238 (1), 47-63 (2001).
- Brand, A. H., Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 118 (2), 401-415 (1993).
- Devraj, K., Guerit, S., Macas, J., Reiss, Y. An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers. Journal of Visualized Experiments. 132, e57038 (2018).
- Fairchild, M. J., Smendziuk, C. M., Tanentzapf, G. A somatic permeability barrier around the germline is essential for Drosophila spermatogenesis. Development. 142 (2), 268-281 (2015).
Cite This Article
Davis, M. J., Talbot, D., Jemc, J. Assay for Blood-brain Barrier Integrity in Drosophila melanogaster. J. Vis. Exp. (151), e60233, doi:10.3791/60233 (2019).