Name: assay for blood-brain barrier integrity in drosophila melanogaster
Uploaded: 2019-09-18T15:00:00
Description: Watch this Scientific Journal Video about Assay for Blood-brain Barrier Integrity in Drosophila melanogaster at JoVE.com

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。

1. 样品收集
<ol><li>胚胎收集
	<ol><li>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。</li>
		<li>预加热苹果汁琼脂板 （表 1） 在 25 °C 过夜. 注：这是适?...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon-glia+interactions+and+the+domain+organization+of+myelinated+axons+requires+neurexin+IV/Caspr/Paranodin.">Axon-glia interactions and the domain organization of myelinated axons requires neurexin IV/Caspr/Paranodin.</a> Neuron. 30 (2), 369-383 (2001).</li><li>Faivre-Sarrailh, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+contactin,+a+homolog+of+vertebrate+contactin,+is+required+for+septate+junction+organization+and+paracellular+barrier+function.">Drosophila contactin, a homolog of vertebrate contactin, is required for septate junction organization and paracellular barrier function.</a> Development. 131 (20), 4931-4942 (2004).</li><li>Salzer, J. L., Brophy, P. J., Peles, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+domains+of+myelinated+axons+in+the+peripheral+nervous+system.">Molecular domains of myelinated axons in the peripheral nervous system.</a> Glia. 56 (14), 1532-1540 (2008).</li><li>von Hilchen, C. M., Bustos, A. E., Giangrande, A., Technau, G. 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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expressional+Profiling+of+Carpet+Glia+in+the+Developing+Drosophila+Eye+Reveals+Its+Molecular+Signature+of+Morphology+Regulators.">Expressional Profiling of Carpet Glia in the Developing Drosophila Eye Reveals Its Molecular Signature of Morphology Regulators.</a> Frontiers in Neuroscience. 13, 244 (2019).</li><li>DeSalvo, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+surface+glia+transcriptome:+evolutionary+conserved+blood-brain+barrier+processes.">The Drosophila surface glia transcriptome: evolutionary conserved blood-brain barrier processes.</a> Frontiers in Neuroscience. 8, 346 (2014).</li><li>. BDSC Cornmeal Food Available from: <a target="_blank" href="https://bdsc.indiana.edu/information/recipes/bloomfood.html">https://bdsc.indiana.edu/information/recipes/bloomfood.html</a> (2017)</li><li>Le, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+family+of+Drosophila+balancer+chromosomes+with+a+w-+dfd-GMR+yellow+fluorescent+protein+marker.">A new family of Drosophila balancer chromosomes with a w- dfd-GMR yellow fluorescent protein marker.</a> Genetics. 174 (4), 2255-2257 (2006).</li><li>Casso, D., Ramirez-Weber, F. A., Kornberg, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP-tagged+balancer+chromosomes+for+Drosophila+melanogaster.">GFP-tagged balancer chromosomes for Drosophila melanogaster.</a> Mechanisms of Development. 88 (2), 229-232 (1999).</li><li>Halfon, M. 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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?...

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。

<table>
	<tbody>
		<tr>
			<td>10 kDa sulforhodamine 101 acid chloride (Texas Red) Dextran</td>
			<td>ThermoFisher Scientific</td>
			<td>D1863</td>
			<td>Dextran should be diluted in autoclaved ddH2O to a concentration of 25 mg/mL.</td>
		</tr>
		<tr>
			<td>20 &#956;L Gel-Loading Pipette Tips</td>
			<td>Eppendorf</td>
			<td>22351656</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol (200 proof)</td>
			<td>Pharmco-Aaper</td>
			<td>11000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Active Dry Yeast</td>
			<td>Red Star</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1423</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP160-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Air Compressor</td>
			<td>DeWalt</td>
			<td>D55140</td>
			<td></td>
		</tr>
		<tr>
			<td>Apple Juice</td>
			<td>Mott&#39;s Natural Apple Juice</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Household Bleach</td>
			<td></td>
			<td>1-5% Hypochlorite</td>
		</tr>
		<tr>
			<td>Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle Plugs</td>
			<td>Fisher Scientific</td>
			<td>AS-277</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainers</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td>Samples imaged using 20x objective (UPlanSApo 20x/ 0.75)</td>
		</tr>
		<tr>
			<td>Cotton-Tipped Applicator</td>
			<td>Puritan</td>
			<td>19-062614</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-Sided Tape 1/2&#34;</td>
			<td>Scotch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Tweezers; Pattern #5; .05 X .01mm Tip</td>
			<td>Roboz</td>
			<td>RS-5015</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly Food Bottles</td>
			<td>Fisher Scientific</td>
			<td>AS-355</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly Food Vials</td>
			<td>Fisher Scientific</td>
			<td>AS-515</td>
			<td></td>
		</tr>
		<tr>
			<td>Foot Pedal</td>
			<td>Treadlite II</td>
			<td>T-91-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Caster</td>
			<td>Bio-Rad</td>
			<td>1704422</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Tray</td>
			<td>Bio-Rad</td>
			<td>1704436</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pipette</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Granulated sugar</td>
			<td></td>
			<td></td>
			<td>Purchased from grocery store.</td>
		</tr>
		<tr>
			<td>Halocarbon Oil</td>
			<td>Lab Scientific, Inc.</td>
			<td>FLY-7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Source</td>
			<td>Schott</td>
			<td>Ace I</td>
			<td></td>
		</tr>
		<tr>
			<td>Manipulator Stand</td>
			<td>World Precision Instruments</td>
			<td>M10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>KITE-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Holder</td>
			<td>World Precision Instruments</td>
			<td>MPH310</td>
			<td></td>
		</tr>
		<tr>
			<td>Nightsea Filter Sets</td>
			<td>Electron Microscopy Science</td>
			<td>SFA-LFS-CY</td>
			<td>For visualization of YFP</td>
		</tr>
		<tr>
			<td>Nightsea Full Adapter System w/ Royal Blue Color Light Head</td>
			<td>Electron Microscopy Science</td>
			<td>SFA-RB</td>
			<td>For visualization of GFP</td>
		</tr>
		<tr>
			<td>Paintbrush</td>
			<td>Simply Simmons</td>
			<td>Chisel Blender #6</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetter</td>
			<td>Fisher Scientific</td>
			<td>13-683C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatic Pump</td>
			<td>World Precision Instruments</td>
			<td>PV830</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Fisher Scientific</td>
			<td>BP363-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Embryo Collection Cages</td>
			<td>Genesee Scientific</td>
			<td>59-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic Anhydrous</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel Base Plate</td>
			<td>World Precision Instruments</td>
			<td>5052</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi 2000</td>
			<td>Used for tissue dissection.</td>
		</tr>
		<tr>
			<td>Stereomicroscope with transmitted light source</td>
			<td>Baytronix</td>
			<td></td>
			<td>Used for injection.</td>
		</tr>
		<tr>
			<td>Tegosept (p-hydroxybenzoic acid, methyl ester)</td>
			<td>Genesee Scientific</td>
			<td>20-258</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td>Nonionic surfactant</td>
		</tr>
		<tr>
			<td>Vial Plugs</td>
			<td>Fisher Scientific</td>
			<td>AS-273</td>
			<td></td>
		</tr>
	</tbody>
</table>

assay for blood-brain barrier integrity in drosophila melanogaster

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。

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...

Watch this Scientific Journal Video about Assay for Blood-brain Barrier Integrity in Drosophila melanogaster at JoVE.com

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

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。

果蝇黑色素血脑屏障完整性分析

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

这种方法可以帮助回答有关血脑屏障在多阶段发育过程中形成和维持的遗传调控关键问题。这种方法也可以适应，以检查其他屏障的渗透性，如肠道上皮在各种生物体。该技术的主要优点是，它允许评估活组织中的血脑屏障功能。此方法的可视化演示至关重要，因为示例操作可能很困难，并且许多步骤需要密切注意细节。对于胚胎收集的地方，至少50个处女与20至25雄性每瓶玉米餐阿加食物，并孵育这些苍蝇一到两天，然后再开始收集。前一天收集，热苹果汁加糖板在25摄氏度过夜。第二天早上，将二氧化碳麻醉苍蝇转移到收集笼中，将预加热的苹果汁加糖板放在笼子的开端上，用一小块酵母膏涂抹。然后用红套将盘子固定到笼子上。为了清除较旧的胚胎，允许苍蝇在25摄氏度的苹果汁加糖盘上产卵一小时。在孵化结束时，将笼子网面向下反转，然后将苍蝇敲击到笼子的底部。用一个新的预热苹果汁加糖板代替苹果汁加糖，用一小块酵母酱涂抹。允许苍蝇在25摄氏度下在新盘子里再产卵一小时。要收集晚期17个胚胎进行注射，用新的预热板代替苹果汁加糖板，用酵母膏涂抹。让苍蝇在25摄氏度的盘子里产卵。一小时后，更换板，用收集的胚胎老化19小时，温度为25摄氏度，使胚胎在成像时20至21小时。在19小时的孵育结束时，从培养箱中取出胚胎收集板，用PbTx覆盖胚板表面。将胚胎的过滤器放在100毫米培养皿中50%的漂白剂中，在室温下偶尔搅拌5分钟，以脱毛。在孵育结束时，在新鲜PBTx中旋转过滤器，使用新的培养皿进行每次洗涤，对胚胎冲洗三次。用PBTx将胚胎清洗到过滤器的一侧，并使用玻璃移液器将胚胎转移到2%的阿加罗斯凝胶板上。用滤纸去除多余的液体。将板面上的六到八个胚胎与右侧的后侧对齐，背侧朝上，并牢固地按下贴在胚胎顶部的幻灯片上的一块双面胶带。然后在室温下干燥胚胎约25分钟，然后用卤化碳油覆盖标本。对于幼虫收集，设置一个十字架与5至10处女雌蝇所需的基因型和一半的雄性所需的基因型在小瓶与玉米粉阿加食品。在25摄氏度的5到7天后，根据基因型，使用钳子轻轻地从小瓶中收集游荡的第三个星幼虫，并用PBS冲洗幼虫，以去除任何卡住的食物。将幼虫转移到苹果汁加糖盘中，必要时进行基因分型，然后再使用画笔将幼虫擦干组织上的幼虫。然后使用钳子将六到八只幼虫转移到用双面胶带准备的幻灯片上。注射前，使用微管拉拔器将毛细管拉成标准针形，用于 D.Melanogaster 注射，并在培养皿中将针头固定在粘土中。对于胚胎注射，使用 20 微升凝胶加载移液器尖端装载准备好的针头，其中 5 微升 10 千吨脱胶素结合到磺胺 101 氯化酸，然后将针头加载到针架中。将针头固定在钢基座的微操纵器中，将注射装置设置为每平方英寸 50 磅，将 5 到 10 毫秒，范围为 10。将幻灯片放在微操纵器舞台上，以 45 度角将针的边缘刷在双面胶带的边缘上，以创建一个稍微倾斜的尖端，以使 10 千吨脱胶器流过开口。泵踩脚踏板，直到染料位于针尖。对齐针头，使其与胚胎平行，并刺穿标本的后端。泵送脚踏板，将两纳米色染料注入试样中。注意用于孵育目的的注射时间，然后继续向下滑动以注入额外的试样。对于幼虫注射，在给幼虫注射220纳米光，与胚胎标本相似之前，使角开口比用于注射胚胎的开口稍大一些，针稍微向下角向标本倾斜。在 10 分钟注射孵化结束时，使用棉尖施用器将油果冻涂在幻灯片上样品的左右两侧，作为垫垫。将盖滑放在顶部，然后将滑轨放在共和显微镜舞台上。选择 20 倍目标，并在整个胚胎深度中成像样本。然后根据公式计算血脑屏障受损的样本百分比。在开始解剖程序之前，使用指甲油在幻灯片上安装两个相距约 0.5 厘米的盖滑，并在 30 分钟孵化结束时将注射的幼虫放入幻灯片上的 PBS 中。使用一对钳子，抓住幼虫身体的一半，用第二对钳子分离幼虫的前部和后部。接下来，使用一对钳子抓住嘴钩的前部区域，并使用第二对钳子将身体壁倒置在第一对钳子的尖端上。大脑和腹神经线将暴露。如果需要，通过切断神经，将大脑和腹间神经线从身体壁分离，然后从滑梯上取下身体壁。用 10 微升 80%甘油覆盖样品。然后在样品上放置盖板，用于成像和成像样品，以确定血脑屏障受损的样本的百分比，如所示。当野生型晚期17胚胎被注射10千达酮脱氧核糖核酸101酸氯化荧光染料时，大德xtran分子被排除在腹神经脐带中，如预期的那样。异质1个突变胚胎表现出完整的血脑屏障，类似于在野生型控制胚胎中观察到的屏障。相比之下，同源1个突变胚胎在血脑屏障的完整性上表现出缺陷，10公斤的脱毒液充斥到腹神经脐带中，表明血脑屏障形成失败。在野生型控制幼虫样本中，10千吨脱毒剂无法穿透血脑屏障，被排除在大脑和腹神经线之外。在尝试此程序时，胚胎的适当老化对于确保样本在血脑屏障形成预期完成的年龄进行检测至关重要。按照这个程序，显示受损血脑屏障的突变体可以使用遗传学、免疫组织化学和显微镜进一步研究，以更好地了解细胞水平上的基因功能。这项技术将使研究人员能够识别对建立和维持血脑屏障至关重要的其他基因。

assay-for-blood-brain-barrier-integrity-in-drosophila-melanogaster

Research

JoVE Journal

Developmental Biology

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

的测定血细胞种群<em&gt;果蝇</em&gt;幼虫

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

饲养果蝇<em&gt;果蝇</em&gt;在无菌和悉生条件

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

协议的可视化类固醇机关及其互动器官在果蝇免疫染色<em&gt;果蝇</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

小鼠血睾丸屏障完整性的一种活体研究方法

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-&#945;-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Methods to Test Endocrine Disruption in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

检测果蝇黑色素的内分泌干扰的方法

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

生成基于人类 iPSC 的血脑屏障芯片

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

使用微计算断层扫描的 果蝇黑色素加斯特 全动物成像

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

来自 黑腹果蝇 第三龄幼虫的活脑外植体成像

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

作者聚焦：研究不对称细胞分裂动力学：果蝇幼虫脑外植体实时成像的方案 

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

黑腹果蝇第三龄幼虫脑的活细胞成像

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...