Name: dissection and live-imaging of the late embryonic drosophila gonad
Uploaded: 2020-10-17T14:00:00.000+00:00
Duration: 9 min 8 s
Description: Watch this Scientific Journal Video about Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad at JoVE.com

我们要感谢Lindsey W. Plasschaert和Justin Sui对该协议的早期开发做出的重大贡献。作者感谢苍蝇界对试剂的慷慨解囊，特别是Ruth Lehmann和Benjamin Lin在出版前赠送的 Nos5'-Lifeact-tdtomato p2a tdkatushka2 Caax nos3'系列。本研究使用了从布卢明顿果蝇库存中心（NIH P40OD018537）获得的股票。这项工作得到了NIH RO1 GM060804，R33AG04791503和R35GM136270（S.D）以及培训补助金T32GM007229（B.W.）和F32GM125123（L.A.）的支持。

1. 解剖前一天的准备
<ol><li>电解磨钨针24 ，使得到的直径约为0.03毫米。将供电电压调节至约14 V，并使用3.3 M NaOH。锐化时间不应超过 1 或 2 分钟。 注意:NaOH具有高度腐蚀性，与皮肤接触会导致灼伤。处理时戴上手套和护目镜，并在通风橱内工作。 注意:使用后，将NaOH储存在聚丙烯猎鹰管中。</li>
	<li>制作准备好的成像介质。在15 mL锥形管中，将4.25 mL施耐德培养基与750μL胎牛血清（FBS，15%终浓度）和27.5μL青霉素-链霉素（0.05 U / μL青霉素，0.05μg/ μL终浓度）混合。将制备的成像培养基储存在4°C。</li>
	<li>制作庚烷胶溶液。将约 0.5 mL 庚烷加入装有约 20 cm 双面胶带的 20 mL 可密封小瓶中。在螺母上摇晃约一小时，或用移液器吸头搅拌，直到达到水和甘油之间的稠度。这种溶解胶水的溶液将持续数天，然后庚烷蒸发。为了清新溶液，再加入0.5mL庚烷和岩石。 注意:可以使用各种比例的庚烷与胶带以及不同的摇摆/搅拌持续时间来制备有效的庚烷胶溶液。上述规格只是准备或清新一种此类适当溶液的建议。</li>
</ol>2. 胚胎采集—解剖前15-17小时
<ol><li>将新鲜酵母糊加入苹果汁琼脂盘25中。通过将成年苍蝇（小于10天大）添加到空食物瓶中来设置胚胎收集笼，并穿孔26个小孔。使用酵母琼脂板盖住收集笼，贴在瓶子上，并将带有板面朝下的笼子放在25°C黑暗培养箱中1小时。 注意:苍蝇必须表达一种转基因，该转基因将荧光标记性腺，例如 ，six4-eGFP::Moesin20，因为胚胎的解剖将在立体荧光显微镜下进行。参见 材料表 ，了解用于标记性腺的基因型列表。</li>
	<li>从培养箱中取出笼子，丢弃第一个集合。用新鲜酵母的琼脂平板代替它，并将笼子放回培养箱中2小时。 注意:第一次胚胎收集用于清除受精，发育中的胚胎的女性，以实现严格时间的第二次胚胎收集。</li>
	<li>从笼子中取出琼脂板，并将其（酵母侧朝上）放在可密封塑料容器内的湿纸巾上。将此潮湿的室置于25°C培养箱中，使胚胎老化14.5小时（最终年龄，产卵后14.5-16.5小时）。 注意:在开始步骤 2.4 之前，请立即完成步骤 3.1 和 3.2。</li>
	<li>从培养箱中取出琼脂板，使用喷瓶，向琼脂板中加入足够的水，用油漆刷轻轻刷洗溶解酵母糊。将胚胎和溶解的酵母冲洗到位于称重船内的小网状筛篮中。用水喷瓶冲洗篮子，直到大多数酵母糊通过网状物过滤。</li>
	<li>从称重船上取出水，然后将篮子放回内部。通过使用喷射瓶将胚胎浸入50%漂白剂溶液中来去皮化胚胎。漂白剂溶液的深度应为3-5毫米。将胚胎浸没在漂白剂中约2分钟，偶尔旋转。 注意:漂白剂具有腐蚀性，会刺激或损害眼睛和呼吸道。处理漂白剂时戴上手套和护目镜。 注意:在此期间，请尽可能完成步骤 3.3。通过将篮子放在体视显微镜下并检查背侧附件的缺失，可以检查脱皮进度。如有必要，将胚胎浸入漂白剂溶液中额外一段时间。</li>
	<li>丢弃漂白剂，并用喷瓶中的水彻底冲洗篮子内的胚胎（约3秒，用纸巾吸网篮;重复2-3次）。</li>
</ol>3. 解剖日准备
<ol><li>在1.5mL Eppendorf管（0.2mg / mL终浓度）中加入30μL胰岛素（10mg / mL）到1，500μL制备的成像培养基（参见步骤1.2）中。充分混合并将管子放在台面上以平衡到室温。</li>
	<li>准备涂有胶水的盖玻片条。<ol>
<li>使用金刚石刀将22 mm x 22 mm盖玻片切割成四个大小相等的条带（图1A）。</li>
		<li>使用镊子拾取一条条，并将总共约30μL庚烷胶溶液涂布到条带的两侧（图1B）。为了获得均匀的胶水残留层，在庚烷蒸发的同时，以各种角度倾斜带材。</li>
		<li>将胶水覆盖的条存放在盖玻片盒的空插槽中;为了保持其粘性，请将条带放置在倾斜但直立的位置，靠在盒子的边缘，以尽量减少与盒子表面的接触（图1C）。关闭盒子，使空气中的颗粒不会涂覆胶水并减轻其粘性。</li>
	</ol>
</li>
	<li>将以下物品放在台面上:6英寸玻璃巴斯德移液器，显微镜载玻片，P200和P1000移液器，具有适当的移液器吸头，Ringer溶液27 （用NaOH调节pH值至7.3）和未覆盖的聚-D-赖氨酸涂层35毫米成像皿。</li>
	<li>将胚胎从网筛篮转移到装有500-750μL庚烷的小手表玻璃杯中。<ol>
<li>用纸巾擦干篮子的侧面和底部。润湿庚烷中的画笔，将画笔触摸到胚胎（疏水性卵黄素膜应粘附在刷毛上），然后将画笔浸回手表玻璃中的庚烷中（胚胎应沉入底部）。 注意:必须尽快执行后续步骤，以防止胚胎变干。胚胎不应暴露在空气中超过20秒。</li>
	</ol>
</li>
	<li>使用巴斯德移液器将胚胎转移到显微镜载玻片上（图1D）。将胚胎缓慢地拉入移液器中，并将其限制在移液器的狭窄部分。将胚胎缓慢移液到载玻片上，使得它们在流到载玻片上之前聚集在移液器的尖端内。将纸巾的角拧成细小的尖端，并吸走载玻片上胚胎上的庚烷。它们将聚集并覆盖较小的区域，从而在下一步中更容易将它们捕获在胶水覆盖的条带上。</li>
	<li>用镊子拿起胶水覆盖的条，轻轻地将其触摸到胚胎（图1E）。将条带放在成像皿中，胚胎侧向放置，并在聚-D-赖氨酸包被的中心外侧。使用镊子将试纸条压在培养皿上，以确保其固定到位。立即用2-3.5 mL林格氏溶液淹没培养皿;首先将胚胎浸入水中以防止它们变干（图1F）。</li>
</ol>4. 解剖
注意:这些步骤必须在立体荧光显微镜下进行。
<ol><li>去除10-15个胚胎的活化。 注意:对于初学者，这可能从两个胚胎到专家的十五个胚胎不等。<ol>
<li>根据肠道形态选择16期胚胎（图2）。在这个阶段，胚胎有三个肠道收缩，产生四个堆叠的肠道片段（图2B，B'）。要开始脱膜化，用钨针刺穿所选胚胎的一端，最好是前部。胚胎可能会从其卵黄素膜中弹出，但如果不是，请将膜从胚胎中剥离出来。 注意:太年轻而无法解剖的胚胎具有非区域化的肠道（图2C）。解剖17期胚胎是可能的，但比解剖16期胚胎更具挑战性，因为角质层在这个阶段开始发育。早期17期胚胎呈现四个相对彼此转移的肠道片段（图2D，D'）。</li>
		<li>将针头钩穿远离性腺的区域的胚胎。将钩住的胚胎转移到培养皿的聚赖氨酸包被区域（从这里开始简称为"盖玻片"），并将其拖到底部直到粘附。重复这些步骤，并沿着聚赖氨酸包被的盖玻片的顶部排列一排脱纤化胚胎（图3A），在下面留出足够的空间用于进一步解剖。 注意:性腺表现为细胞的小球形聚集体，应发出明亮的荧光，具体取决于使用的特定标记物和转基因拷贝数。在这个阶段，性腺位于A5节的横向位置，距离前部约70%-80%的胚胎长度。在整个解剖方案中，组织可能会粘在针头上。要清除针头上的碎屑，请将其抬高到Ringer溶液的表面上方。去玻璃化可以不整洁，只要性腺本身受到尽可能少的干扰。</li>
	</ol></li>
	<li>将性腺从胚胎中取出并放到培养皿上（图3C，D）。<ol>
<li>首先，将胚胎暴露其内部（图3C）。从胚胎的中心切开胚胎，将针向后移动，在性腺之间。梳理一些内部组织，因为这会比外部角质层更好地粘附在盘子上。组织的粘性将使下一步的操作能够揭示性腺并使其粘附在盖玻片上。 注意:如果纸巾没有粘在培养皿上，请尝试将其哄到涂层盖玻片的未受污染区域;盖玻片的外部区域会特别粘。如步骤4.1.2所述，只要性腺毫发无损，解剖操作是否导致胚胎胴体受损并不重要。</li>
		<li>用针头切开性腺，直到一块组织（包括性腺）与剩余的胴体分离。用针头将该组织拉到涂有盖纸的新鲜区域，并将其哄到底部，直到它粘附在培养皿上。 注意:对于性腺本身直接附着在盖玻片上而不是通过上覆组织间接附着的情况，将实现最佳成像。因此，当组织被引导离开胴体时，尝试让性腺首先接触覆盖物，而不是外来组织。</li>
		<li>通过用针轻轻地将粘附组织从性腺上拖走，从性腺中取出尽可能多的周围组织（图3D）。避免直接触摸性腺，否则会损坏性腺（图4E）。为确保性腺充分粘附在培养皿上，请围绕性腺以轻柔的圆周运动移动针头 - 如果它移动，将针头触摸剩余的粘附组织，并将性腺引导至粘性覆盖物的新鲜区域。重复此过程，直到没有可检测到的性腺运动。</li>
		<li>返回胚胎胴体并解剖出第二个性腺。在尽可能多的胚胎上重复此过程，但不要超过25分钟。组织活力将在超过约40-45分钟后在Ringer溶液中受损。</li>
	</ol></li>
	<li>解剖完成后，使用不可磨灭的标记在成像盘的外缘添加套准标记，以记录解剖过程中的方向。</li>
	<li>从盘子中取出涂有胶水的条。<ol>
<li>轻轻地将一对镊子的底部插脚插入条带下方，扣住并缓慢向上倾斜，以将其从培养皿中释放出来，并尽可能少地晃动Ringer溶液，以尽量减少对粘附性腺的干扰。 注意:条带应倾斜远离解剖的性腺。</li>
	</ol></li>
	<li>轻轻地将培养皿带到成像显微镜上，以避免铃声溶液晃动。</li>
</ol>5. 成像
<ol><li>将成像皿放在载物台支架中，使用套准标记将培养皿放置在解剖过程中的近似方向。使用明场显微镜和低功率（~10倍）物镜，识别并聚焦粘附在盖玻片上的任何组织。切换目镜设置以显示荧光，并使用双目目镜系统地扫描培养皿，在成像软件中标记每个性腺的位置（参见 材料表）。 注意:在标记位置之前，请确保性腺在视野中居中。</li>
	<li>轻轻地取下整个载物台支架组件，将成像盘放在支架中，然后将组件放在工作台上。</li>
	<li>用含有胰岛素的制备成像培养基替换Ringer的溶液（参见步骤1.2和3.1）。<ol>
<li>使用P1000从成像皿的内侧上壁架上取下所有Ringer溶液（不要从中央盖玻片区域移液Ringer）。接下来，切换到P200，并将其尖端放在中央区域剩余铃声的表面下方。小心地去除50-100μL林格氏菌;不要删除整个解决方案。</li>
		<li>抽取约200μL成像培养基，然后再次将P200尖端放在剩余Ringer的表面下方，慢慢加入该成像培养基。接下来，将剩余的成像介质（约1，300μL）加入培养皿中，从上壁架的最外缘开始。移液时，向流体的中央圆顶移动，并最终通过将移液器尖端刷过它们来合并两者。将盖子放在盘子上。 注意:成像介质应覆盖培养皿的整个内径，深度约为2 mm，以防止蒸发（图4D）。</li>
	</ol>
</li>
	<li>将显微镜切换到更高功率的物镜（63x，1.2 NA），根据所使用的物镜（浸入液类型和物镜所需的折射率）应用适当的浸入液，然后更换载物台支架组件。使用明场显微镜聚焦在成像皿底部的组织上。 注意:如果载物台未移动，则一旦物镜聚焦，最后一个性腺应进入视野。</li>
	<li>逐步浏览每个标记的性腺位置，并根据需要调整该位置。根据图像清晰度，性腺性别等选择要成像的性腺。自定义成像设置（多通道、曝光时间、激光强度、Z 系列增量、具有适当间隔的延时摄影等）。开始成像。 注意:雄性性腺可以通过存在一个生态位和在性腺的另一端，一簇称为雄性特异性体细胞的小型，高度圆形的体细胞来识别，称为雄性特异性体细胞性腺前体（msSGP）。如果使用 六4-eGFP::moesin荧光标记物，则利基细胞是性腺中第二亮的细胞，最亮的是msSGP。在这个阶段，雌性性腺既没有利基，也没有msSGP。</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+live+imaging+of+stem+cells+in+drosophila+testis.">Time-lapse live imaging of stem cells in drosophila testis.</a> Current Protocols in Stem Cell Biology. 0331, 1-9 (2009).</li><li>Anllo, L., Plasschaert, L. W., Sui, J., DiNardo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+reveals+hub+cell+assembly+and+compaction+dynamics+during+morphogenesis+of+the+Drosophila+testis+niche.">Live imaging reveals hub cell assembly and compaction dynamics during morphogenesis of the Drosophila testis niche.</a> Developmental Biology. 446, 102-118 (2019).</li><li>Rebecca Sheng, X., Matunis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+the+Drosophila+spermatogonial+stem+cell+niche+reveals+novel+mechanisms+regulating+germline+stem+cell+output.">Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output.</a> Development. 138, 3367-3376 (2011).</li><li>Aboim, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developpement+embryonnaire+et+post-embryonnaire+des+gonades+normales+et+agametiques+de+Drosophila+melanogaster.">Developpement embryonnaire et post-embryonnaire des gonades normales et agametiques de Drosophila melanogaster.</a> Revue Suisse De Zoologie. 52, 53 (1945).</li><li>Sano, H., 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+actin+regulator+ENABLED+regulates+cell+shape+and+orientation+during+gonad+morphogenesis.">The Drosophila actin regulator ENABLED regulates cell shape and orientation during gonad morphogenesis.</a> PLoS One. 7, (2012).</li><li>Clark, I. B. N., Jarman, A. P., Finnegan, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+Drosophila+gonad+formation+reveals+roles+for+Six4+in+regulating+germline+and+somatic+cell+migration.">Live imaging of Drosophila gonad formation reveals roles for Six4 in regulating germline and somatic cell migration.</a> BMC Devlopmental Biology. 7, 1-9 (2007).</li><li>Sheng, X. R., Brawley, C. M., Matunis, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedifferentiating+spermatogonia+outcompete+somatic+stem+cells+for+niche+occupancy+in+the+Drosophila+testis.">Dedifferentiating spermatogonia outcompete somatic stem cells for niche occupancy in the Drosophila testis.</a> Cell Stem Cell. 5, 191-203 (2009).</li><li>Tanentzapf, G., Devenport, D., Godt, D., Brown, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Europe+PMC+Funders+Group+integrin-dependent+anchoring+of+a+stem+cell+niche.">Europe PMC Funders Group integrin-dependent anchoring of a stem cell niche.</a> Nature Cell Biology. 9, 1413-1418 (2007).</li><li>Brady, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+making+very+fine,+durable+dissecting+needles+by+sharpening+tungsten+wire+electrolytically+a+motorized+molluscicide+dispenser.">A simple technique for making very fine, durable dissecting needles by sharpening tungsten wire electrolytically a motorized molluscicide dispenser.</a> Bulletin of the World Health Organization. , 105-106 (1960).</li><li>Featherstone, D. E., Chen, K., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvesting+and+preparing+Drosophila+embryos+for+electrophysiological+recording+and+other+procedures.">Harvesting and preparing Drosophila embryos for electrophysiological recording and other procedures.</a> Journal of Visualized Experiments: JoVE. , (2009).</li><li>JoVE. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+and+Larva+Harvesting+and+Preparation.">Embryo and Larva Harvesting and Preparation.</a> JoVE Science Education Database. , (2020).</li><li>Cold Spring Harbor Protocols. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ringer's+solution+for+Drosophila.">Ringer's solution for Drosophila.</a> Cold Spring Harbor Protocols. , (2011).</li><li>Schuh, M., Lehner, C. F., Heidmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+Drosophila+CID/CENP-A+and+CENP-C+into+Centromeres+during+Early+Embryonic+Anaphase.">Incorporation of Drosophila CID/CENP-A and CENP-C into Centromeres during Early Embryonic Anaphase.</a> Current Biology. 17, 237-243 (2007).</li><li>Lin, B., Luo, J., Lehmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collectively+stabilizing+and+orienting+posterior+migratory+forces+disperses+cell+clusters+in+vivo.">Collectively stabilizing and orienting posterior migratory forces disperses cell clusters in vivo.</a> Nature Communications. 11, 1-16 (2020).</li><li>Prasad, M., Jang, A. C. C., Starz-Gaiano, M., Melani, M., Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+culturing+drosophila+melanogaster+stage+9+egg+chambers+for+live+imaging.">A protocol for culturing drosophila melanogaster stage 9 egg chambers for live imaging.</a> Nature Protocols. 2, 2467-2473 (2007).</li><li>Gabay, L., Lowell, S., Rubin, L. L., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deregulation+of+dorsoventral+patterning+by+FGF+confers+trilineage+differentiation+capacity+on+CNS+stem+cells+in+vitro.">Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro.</a> Neuron. 40, 485-499 (2003).</li><li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126, 677-689 (2006).</li></ol>

作者没有什么可透露的。

在性腺发生期间，胚胎性腺，特别是雄性性腺15内的干细胞位，经历快速的形态变化。这些动态变化背后的发育机制最好通过实时成像技术来理解。然而，在胚胎阶段17，性腺的 体内 成像由于大规模肌肉收缩的发作而变得不可能17。通过该协议，我们提供了一种成功的替代方案:将性腺直接解剖到成像盘上进行 体外 实时成像。该协议是完成晚期胚?...

黑腹果蝇睾丸已成为我们理解许多动态细胞过程的范式。该模型的研究揭示了干细胞分裂调控1，2，3，生殖细胞发育4，5，piRNA生物学6，7，8和利基干细胞信号传导事件9，10，11，12，13。该模型是有利的，因为它在遗传上是可处理的14，15并且是少数我们可以在其自然环境中实时成像干细胞3，16，17，18的模型之一。然而，该模型的活体成像仅限于成体组织和早期胚胎阶段，在我们对晚期胚胎性腺动力学的知识中留下了空白，这是生态位首次形成并开始发挥作用的精确阶段。
晚期胚胎性腺是一个球体，由前部的体细胞位细胞组成，生殖细胞由体细胞在更后部区域19中形成。该器官可以在体内实时成像，直到早期胚胎阶段1717，20，21。由于开始大规模肌肉收缩，可避免进一步影像学检查。这些收缩非常严重，以至于它们将性腺推出成像框架，并且这种运动无法用成像软件进行校正。我们的实验室有兴趣揭示生态位形成的机制，这发生在这个难以捉摸的实时成像时期。因此，我们生成了一种 离体 方法，从胚胎阶段16开始对性腺进行实时成像，从而促进了在这个关键时期的性腺发育期间细胞动力学的研究。我们实验室先前的工作表明，这种 离体 成像忠实地概括了 体内 性腺发育17。 这种技术是果 蝇 胚胎性腺的第一种，也是唯一一种。
在这里，我们介绍了在胚胎晚期期对性腺进行 体外 活体成像所需的解剖方案。该方案可以与药物治疗相结合，或对性腺内特定细胞系进行转基因操作。使用这种技术，我们已经成功地成像了干细胞生态位形成的步骤17。因此，这种成像方法对于干细胞生物学领域是有帮助的，因为它将能够在其自然环境中实时可视化生态位形成的初始阶段15，17。虽然这种方法有利于干细胞生物学领域，但它也适用于可视化在该发育时间点期间性腺中发生的任何动态过程，包括细胞重排22，细胞粘附2，12，23和细胞迁移23。因此，这种解剖方案将增强我们对许多基本细胞生物学过程的理解。

<table><tbody><tr><td>Alfa Aesar Tungsten wire</td><td>Fisher Scientific</td><td>AA10408G6</td><td>0.25mm (0.01 in.) dia., 99.95% (metals basis)</td></tr><tr><td>&lt;em&gt;D. melanogaster:&lt;/em&gt; His2Av::mRFP1</td><td>Bloomington Drosophila Stock Center (BDSC)</td><td>FBtp0056035</td><td>Schuh, Lehner, &amp;amp; Heidmann, Current Biology, 2007</td></tr><tr><td>&lt;em&gt;D. melanogaster: nos&lt;/em&gt;-lifeact::tdtomato</td><td>Gift from Ruth Lehmann Lab</td><td></td><td>Lin, Luo, &amp;amp; Lehmann, Nature Communications, 2020: nos5&#039;- Lifeact-tdtomato p2a tdkatushka2 Caax nos3&#039;</td></tr><tr><td>&lt;em&gt;D. melanogaster: P-Dsix4-eGFP::Moesin&lt;/em&gt;</td><td></td><td>FBtp0083398</td><td>Sano et al., PLoS One, 2012</td></tr><tr><td>Diamond-tipped knife</td><td></td><td></td><td></td></tr><tr><td>Double-sided tape</td><td>Scotch</td><td>665</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>GIBCO</td><td>10082</td><td></td></tr><tr><td>Imaging dish</td><td>MatTek</td><td>P35GC-1.5-14-C</td><td></td></tr><tr><td>Imaging software</td><td>Molecular Devices</td><td></td><td>MetaMorph Microscopy Automation and Image Analysis Software v7.8.4.0</td></tr><tr><td>Insulin, bovine</td><td>Sigma</td><td>l0516</td><td>Store aliquots at 4 &amp;deg;C</td></tr><tr><td>Needle holder</td><td>Fisher Scientific</td><td>08-955</td><td></td></tr><tr><td>Nytex basket</td><td></td><td></td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Corning</td><td>30-002-Cl</td><td></td></tr><tr><td>Ringer&#039;s solution</td><td></td><td></td><td>2 mM MgCl2, 2 mM CaCl2, 130 mM NaCl, 5mM KCl, 36 mM Sucrose, 5mM Hepe&amp;rsquo;s Buffer; adjusted with NaOH until pH of 7.3 is achieved</td></tr><tr><td>Schneider&#039;s Insect Media</td><td>GIBCO</td><td>21720-024</td><td></td></tr></tbody></table>

dissection and live-imaging of the late embryonic drosophila gonad

黑腹果蝇雄性胚胎性腺是研究发育生物学各个方面的有利模型，包括但不限于生殖细胞发育，piRNA生物学和生态位形成。在这里，我们提出了一种解剖技术，用于在体内实时成像非常无效的时期对性腺进行活体成像。该协议概述了如何将胚胎转移到成像皿中，选择适当分期的雄性胚胎，并在保持其结构完整性的同时从其周围组织中解剖性腺。解剖后，可以使用共聚焦显微镜对性腺进行成像，以可视化动态细胞过程。解剖过程需要精确的时间和灵活性，但我们提供了有关如何防止常见错误以及如何克服这些挑战的见解。据我们所知，这是果蝇胚胎性腺的第一个解剖方案，并且将允许在其他无法进入的时间窗口进行现场成像。该技术可以与药理学或细胞类型的特异性转基因操作相结合，以研究在其自然性腺环境中细胞内或细胞之间发生的任何动态过程。

我们在 图1中说明了成像皿的制备，如"解剖日准备"中所述。这些方法最终应导致水合良好的胚胎粘附在盖滑条上，该衬裙暂时固定在培养皿的底部并浸没在Ringer的溶液中（图1F）。金刚石刀允许将22 x 22 mm的盖玻片干净地切成三到四条较小的条（图1A）。在用镊子处理这些条时，我们使用移液器转移足够的庚烷胶来涂覆这些条带，?...

Watch this Scientific Journal Video about Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad at JoVE.com

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

在这里，我们提供了对晚期胚胎 果蝇 雄性性腺进行现场成像所需的解剖方案。该协议将允许在正常条件下或在转基因或药理学操作之后观察动态细胞过程。

晚期胚胎 果蝇 性腺的解剖和活体成像

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

dissection-and-live-imaging-of-the-late-embryonic-drosophila-gonad

Research

JoVE Journal

Developmental Biology

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

3.2K 次观看.  University of Pennsylvania. 在这里，我们提供了对晚期胚胎 果蝇 雄性性腺进行现场成像所需的解剖方案。该协议将允许在正常条件下或在转基因或药理学操作之后观察动态细胞过程。

视频: Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

科研

发育生物学

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

长期的实时成像<em&gt;果蝇</em&gt;眼睛光盘

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

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.

住在果蝇眼睛成虫光盘胶质细胞迁移成像

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

生物学

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

为介观荧光体层摄影术在体内成像的发展果蝇</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

现场剖析果蝇胚胎抗体染色筛选突变体集合的简化方法

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. 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 ...

Real-Time Imaging of Germline Stem Cells in Drosophila Ex Vivo Germaria Culture

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs&#39; asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to ...

晚期胚胎果蝇性腺的解剖和活体成像

摘要

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实时成像

长期的实时成像

住在果蝇眼睛成虫光盘胶质细胞迁移成像

为介观荧光体层摄影术在体内成像的发展果蝇

现场剖析果蝇胚胎抗体染色筛选突变体集合的简化方法

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

黑腹果蝇第三龄幼虫脑的活细胞成像

黑腹果蝇第三龄幼虫脑的活细胞成像

黑腹果蝇第三龄幼虫脑的活细胞成像

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

晚期胚胎 果蝇 性腺的解剖和活体成像

摘要

探索更多视频

此视频中的章节

实时成像

长期的实时成像

住在果蝇眼睛成虫光盘胶质细胞迁移成像

为介观荧光体层摄影术在体内成像的发展果蝇

现场剖析果蝇胚胎抗体染色筛选突变体集合的简化方法

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

黑腹果蝇第三龄幼虫脑的活细胞成像

黑腹果蝇第三龄幼虫脑的活细胞成像

黑腹果蝇第三龄幼虫脑的活细胞成像

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

晚期胚胎果蝇性腺的解剖和活体成像