该UMS3444生物科学，里昂，法国PLATIM和Droso - 工具设施。 BM的研究是由基金会拨款支持的倒拉RECHERCHEMédicale来自法国国家科学研究中心（ATIP）和ANR-12-BSV1-0019-01。帕金森病是由视网膜法国协会和里昂高等师范学院（法国）的支持。 CL是由La法甲国立CONTRE乐癌症协会的支持。

1。番茄/ GFP-FLP / FRT线的交叉苍蝇携带突变克隆的产生需要一个FRT携带突变系和相应的番茄/ GFP-FLP / FRT飞线之间的单交。 这项工作动用的首次登记税，重组突变BruinFly集合（ <a href="http://www.bruinfly.ucla.edu" target="_blank">http://www.bruinfly.ucla.edu</a> ）。四个番茄/ GFP-FLP / FRT线可以使用，携带FRT甙Rh1-tdtomatoninaC 2号的每个武器和第三染色体结合flipase源（EY-FLP）和GFP在所有外的PR（甙Rh1-GAL4 UAS表达-GFP）20： - 2L手臂：行＃43345，P {RY [+ T7.2] = RH1-GAL4} 1，P {RY [+ T7.2] = EY-FLP.N} 2，W [*]，P {白[+ MC] = ninaE-tdTomato-ninaC} 2L p {RY [+ T7.2] = neoFRT} 40A，P {W [+ MC] = UAS-GFP-ninaC} 3 - 2R手臂：行＃43346，P {RY [+ T7.2] = RH1-GAL4} 1，P {RY [+ T7.2] = EY-FLP.N} 2，W [*]，P {RY [+ T7.2] = neoFRT} 42D P{W [+ MC] = ninaE-tdTomato-ninaC} 2R，P {W [+ MC] = UAS-GFP-ninaC} 3 - 3L手臂：行＃43347，P {RY [+ T7.2] = RH1-GAL4} 1，P {RY [+ T7.2] = EY-FLP.N} 2，W [*]，P {白[+ MC] = UAS-GFP-ninaC} 2，P {W [+ MC] = ninaE-tdTomato-ninaC} 3L p {RY [+ T7.2] = neoFRT} 80B/TM6B，铽[1] - 3R手臂：行＃43348，P {RY [+ T7.2] = RH1-GAL4} 1，P {RY [+ T7.2] = EY-FLP.N} 2，W [*]，P {白[+ MC] = UAS-GFP-ninaC} 2，P {RY [+ T7.2] = neoFRT} 82B p {W [+ MC] = ninaE-tdTomato-ninaC} 3R/TM6B，铽[1] 这四条线可在布卢明顿果蝇库存中心。它们在显影眼有丝分裂克隆21的产生提供FLP的来源。与野生型染色体携带的FRT序列和RH1-tdTomato ninaC构建编码红色荧光蛋白tdTomato，作为标记的野生型和杂合的外积比率。在这种结构中，ninaC的P174亚型的最后41个氨基酸（既不失活，也不激活C）基因是AP悬浮于帧到tdTomato序列的C-末端。在P174 ninaC异构体的C末端负责蛋白22的rhabdomeral本地化和许可证使用角膜术中和红色荧光的PR更好的实时可视化。该RH1启动子是最小的234个基点（-152 +82）RH1的启动子。绿色荧光标记的GFP是所有的外积比率表示，由于RH1-GAL4（3kb的启动子）和UAS-GFP ninaC构造的存在。一只苍蝇股票携带突变上的x染色体上首次登记税（FRT-x）和番茄/ GFP-FLP / FRT线之间杂交产生的后代具有下列基因型（突变对2L的例子）：RH1-GAL4，EY-FLP ; FRT40A，RH1-tdTomato ninaC / FRT40A-X; UAS-GFP ninaC。在这些苍蝇，通过有丝分裂重组的显影眼圆盘产生，分化，以产生纯合的突变体的PR的克隆（ 图2A）的纯合突变体的细胞。智人合子突变体细胞可被识别，因为它们只表达绿色荧光蛋白（GFP），而周围的野生型和杂合的细胞同时表达tdTomato和GFP（ 图2B和B“）。 2。准备果蝇的感光可视化对于PR可视化的角膜中和技术，固定苍蝇到琼脂糖平板上。 <ol><li>准备一个洗瓶子装满4℃的蒸馏水，将其放在冰上。 </li><li>通过加热，在微波溶于水常规琼脂糖（在100ml水中1.2-1.5％琼脂糖）。将烧瓶在水浴中在55℃，并允许将琼脂糖溶​​液冷却至该温度。保持琼脂糖在55℃备用。 </li><li>麻醉蝇与CO 2为至少1分钟。 </li><li>倒入温热的琼脂糖溶液（55℃）放入培养皿（35×10毫米，1.37 x 0.39英寸），并将immediately的麻醉果蝇到琼脂糖。对于初学者，开始与10苍蝇每培养皿是合理的。大陪替氏培养皿（60×15毫米，2.362×0.59英寸的）也可以使用。 </li><li>在解剖显微镜下，定向果蝇在其一侧与钳，推一翼入琼脂糖，使得主体的翼和半嵌入在琼脂糖（ 图3A-3A“”）。棒的另一翼上的琼脂糖的表面上。注意：如果苍蝇不深深嵌入足够的琼脂糖，飞将免费为公关的成像过程中移动它的头，这会导致图像模糊。眼睛的取向也将受到干扰。它可能难以沉浸果蝇到琼脂糖，由于琼脂糖的任一浓度或温度。这是比较容易嵌入苍蝇更浓的琼脂糖，但如果琼脂糖过于集中，苍蝇可能下沉。如果琼脂糖过凉，也可能是二fficult嵌入到飞，但过高的温度可能会损伤角膜。 </li><li>把培养皿上的冰，让琼脂糖凝固。 </li><li>用钳子，定位头，使得一只眼睛暴露于浸没物镜（ 图3A&#39;和3A“”）。通常，眼可认为在解剖显微镜下很好地定向时pseudopupil（可视化为一个黑点）是在眼睛的中间。眼的最佳取向最大化ommatidial字段中的PR集中的宽度。取向步骤的目的是要找到眼睛的区域与集中的PR的最宽领域中，通常在眼的中心。这个步骤也用于除去任何腿或aristae覆盖在眼睛和防止其可视化是有用的。 </li><li>覆盖用冰冷的水飞，离开培养皿置于冰上，直到可视化。冰冷的水保持麻醉苍蝇。 </li></ol> 3。可视化在显微镜下光感受器对于永久居民的通过角膜的可视化，这个协议使用一个堂堂正正的显微镜配备水目标具有长工作距离（W N-Achroplan 40X/0.75）。 <ol><li>放置在培养皿盖，用冰冷的水对显微镜的阶段载玻片（ 图公元前3）。陪替氏培养皿可粘在载玻片上，使其能够与测微螺钉将其移动大约顺利。 </li><li>暴跌浸泡的目的在培养皿（ 图3B&#39;-3C&#39;）的水。定位在激发光束下飞散的头部（开始与GFP过滤器，例如）。将舞台上下，直到收敛束对眼睛。当眼睛处于合适的水平，它往往反映的激发光。 </li><li>看看通过目镜的荧光。当眼睛被定位在视场的中心，着眼下面的角膜可视化荧光光感受器。 </li></ol>注1：当通过目镜看，这可能是不易区别的身体部位的苍蝇，腹部尤其远侧部分和头部的，例如。在这种情况下，你应该直接看舞台上重新定位果蝇。 注2：古典荧光或共聚焦显微镜也可使用。共聚焦显微镜给人以较少的背景和针对性的减贫战略的一个更广泛的领域比传统的显微镜图像。一个例子设置是一个LSM510共聚焦显微镜（蔡司）与40X水的目标。更好的结果可以通过打开的共聚焦显微镜更广泛的针孔比通常建议的目标来获得。例如，使用一个204的值，而不是默认的98针孔的光圈。 注3：更高级别的眼球W +色素沉着减少背景荧光。 </p&gt; 4。感光细胞的时间过程可视化按照相同的眼内经过一段时间的个人PR的命运时需要小心。 <ol><li>减少琼脂糖的温度提高到45℃，以最大限度地提高果蝇存活。琼脂糖很快固化在该温度下。只有一个果蝇应每培养皿中被使用，以确保在苍蝇的观察不会被混 ​​合起来。 </li><li>系统定位在同一侧的苍蝇，用于查看相同的眼睛。这可以通过服用麻醉蝇从它的小瓶被一个翼和放置果蝇上的琼脂糖来实现。始终定位眼睛以同样的方式，如果可能，因此很容易发现，之前已经观察公关克隆。 </li><li>确定克隆它们的形状的基础上的共焦显微镜下。视野往往是毗邻感兴趣的克隆。在苏通道的情况下，眼睛应该在水中被重新定向，以居中的视场在感兴趣的克隆的基础上，眼睛的极性（ 图4）。 </li></ol> 5。可视化后恢复苍蝇<ol><li>从培养皿中除去水。 </li><li>轻轻一拉果蝇用钳琼脂糖。 </li><li>干果蝇的组织。 </li><li>返回果蝇到小瓶中，并确保它不被卡住的食品。宠物苍蝇来轮在25°C。 </li></ol>

<ol>
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作者什么都没有透露。

果蝇眼睛已被广泛用于破译的信号转导途径调节发育，增殖和存活。在90年代初期，大量的遗传筛选的进行，以确定在公关发展25-27的初始阶段所需要的途径。遗传筛选的效率是由有丝分裂重组技术，这使得它可以在镶嵌克隆21测试功能丧失的突变大大增加。因此，胚胎致死突变的作用，可以在系统中苍蝇的眼睛纯合突变克隆是杂合子，否则测试。大多数马赛克放映都集中在早期公关招聘，分化，轴突投射或在发展中的幼虫或蛹10,25-31发生形态。迄今为止，只有一个拼接屏已经研究了调节成年公关功能的机制，透过electroretin监控视觉反应ogram录音32。与番茄/ GFP-FLP / FRT和方法在活突变的动物进行时间过程分析的可能性，我们设计了用于识别调节成年PR可行性的因素的新方法和功能20,24。 番茄/ GFP-FLP / FRT方法比树脂包埋眼中的经典组织学切片几大优势。首先，这种方法是更快，更便宜和更容易比组织学方法来执行，只要配备适当的水浸渍客观荧光显微镜是可用的（ 见表试剂和设备 ）。其次，番茄/ GFP-FLP / FRT方法可以与转基因表达结合使用，如P的表达式（UAS，W +），携带一个微型白色转基因。相反，组织切片，其中W +是用于克隆的检测，在番茄/ GFP-FLP / FRT系统，P（UAS，瓦特+）不与克隆ð干扰etection基于番茄荧光蛋白。使用P（UAS-FATP，W +）转基因果蝇，我们能够以可视化的FATP突变的PR救援（ 图7）。第三，所有类型的克隆分析，包括基于番茄/ GFP-FLP / FRT，是失去永久居民的基因型的模糊性的一个重要缺陷。事实上，它可能是不清楚的一个PR是否是因为或者由于细胞非自主的效果，特别是在突变克隆的边界缺乏特定基因的功能缺失。番茄/ GFP-FLP / FRT方法通过使得可以遵循野生型和突变体的PR在同一动物在数周期间得到解决这个问题对于变性。我们能够遵循的动力学个人收受的命运分析不同FATP突变果蝇上（ 图6）。相同的克隆可以在给定的动物在不同的时间被识别的周围野生型克隆的精确形状。我们能够清晰展示LY所有丢失的PR是突变的FATP，表明FATP突变体中的PR的作用是细胞自主性。因此，通过监测的PRs在动力学分析的损失，因此能够确定的PRs中的成人发病变性模型的基因型。最后，将番茄/ GFP-FLP / FRT方法已经被证明是非常强大的用于调节因子建立的PCP 20的识别。中进球了大量的马赛克小眼的极性表型，而不需要部分的可能性，有利于快速测定的公关要求建立的PCP。尽管如此，公关完整性精细分析需要传统切片后相衬或电子显微术。 总之，番茄/ GFP-FLP / FRT方法开辟了高效的马赛克筛选新的可能性，以确定调节因子的PR发育过程和成年PR的功能，如在相UAL响应和可行性。

果蝇视网膜是由大约800 ommatidial单元（ 图1A），它被精确地组织，具有明确定义的极性的轴线（ 图1B）。每个单元包含20个单元：8感光细胞（PRS）和12个辅助细胞，包括视锥细胞，色素细胞和猪鬃细胞1,2。两个班的公关来区分的视紫红质，他们表达型（RH）的基础上。六个外的PR（R1-6）表达Rh1的和被安排在一个梯形图案（ 图1C）。在梯形的中心，两个内积比率（R7/R8）表示四种可能类型的视紫红质（Rh3的，RH4，RH5或RH6），组织，使得R7在于R8 3的顶部上。 2500多个基因参与了果蝇眼睛4，这已被证明是非常强大的模型的过程很宽面板，包括眼睛德韦研究的形态讲习班中，公关招聘，分化，平面细胞极性，形态，存活，凋亡和视觉传导5-9。 研究人员工作在果蝇眼睛都，多年来，开发技术成像在视网膜和开展系统性的遗传筛选。最简单的方法对图像中的成人视网膜是看角膜在一个固定的动物（ 图1A）。角膜的结构，可以精确地通过扫描电子显微镜观察，并用在大规模遗传筛选由URCFG财团（ <a href="http://www.bruinfly.ucla.edu" target="_blank">http://www.bruinfly.ucla.edu</a> ）10。这是一种非常有效的方法用于可视化在角膜结构全局形态学变化，如眼睛的粗糙度或光泽度，由突变引起的常在控制基因在发育或细胞活性的早期步骤。然而，角膜全球可视化是不是sufficient识别调节公关rhabdomere形态或者成人公关活力和功能的因素。这种分析需要进行更彻底的调查，公关诚信的基础上，视网膜11-13切半薄树脂切片相衬显微镜。此技术适用于镶嵌的分析，其中突变体的PR可以通过其缺乏红色素14,15来识别。 花叶突变体克隆，也可以显现在蛹或成年视网膜的整个贴装夹层，其缺少绿色荧光蛋白（GFP）的信号上荧光显微镜16,17的基础上。这两种技术是非常有用的，但两者都是劳力和费时，因此不适合大规模筛选。我们和其他人已经开发利用的角膜中和技术生产的PR荧光蛋白18,19的成像，便于快速公关分析。利用这种技术，突变体克隆可识别的红色（W +）颜料在镶嵌成年果蝇克隆的自体荧光的基础上。然而，这种方法不提供解决细胞自主性所需要的单细胞分辨率问题19。我们通过开发番茄/ GFP-FLP / FRT方法，它结合了荧光蛋白的角膜中和与有丝分裂重组20的成像克服了这个问题。这种方法允许高通量，快速和准确地识别突变体的PR在镶嵌克隆，在单细胞分辨率（ <a href="http://www.ens-lyon.fr/LBMC/ApoDrosoDatabase/" target="_blank">http://www.ens-lyon.fr/LBMC/ApoDrosoDatabase/</a> ）。它适用于在动力学分析使用，对于居住在果蝇以下个人永久居民在一个星期内。我们在这里描述的番茄/ GFP-FLP / FRT方法和技巧及其在镶嵌的眼睛简单，动力学分析使用。

<table border="1">
	<tbody>
		<tr>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				Reagent</td>
		</tr>
		<tr>
			<td>
				Agarose</td>
			<td>
				Euromedex</td>
			<td>
				D5-E</td>
			<td>
				Regular agarose is used to immobilize the flies</td>
		</tr>
		<tr>
			<td>
				Petri dish</td>
			<td>
				BD Falcon</td>
			<td>
				353001</td>
			<td>
				35 x 10 mm, 1.37 x 0.39 inch</td>
		</tr>
		<tr>
			<td>
				Cold-ice water</td>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				To maintain the flies anesthetized</td>
		</tr>
		<tr>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				[HEADER]</td>
		</tr>
		<tr>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				Equipment</td>
		</tr>
		<tr>
			<td>
				Dissecting microscope</td>
			<td>
				Dutcher</td>
			<td>
				SMZ645</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Water Bath</td>
			<td>
				Julabo</td>
			<td>
				9550102</td>
			<td>
				To keep the agarose solution at warm temperature</td>
		</tr>
		<tr>
			<td>
				40X Water objective</td>
			<td>
				Zeiss</td>
			<td>
				420967-9900-000</td>
			<td>
				Water dipping objective for the confocal microscope</td>
		</tr>
	</tbody>
</table>

	&nbsp;

the tomato/gfp-flp/frt method for live imaging of mosaic adult drosophila photoreceptor cells

果蝇眼睛被广泛用作模型的开发和神经元变性的研究。凭借强大的有丝分裂重组技术的基础上，克隆分析典雅的遗传筛选，导致参与眼睛发育和光感受器（PR）的分化在幼虫阶段信号途径的鉴定。我们在这里描述的番茄/ GFP-FLP / FRT方法，该方法可用于在活的成年果蝇的眼睛快速克隆分析。荧光灯的光感受器细胞进行成像与角膜中和技术，对视网膜与flipase-介导的重组产生的马赛克克隆。该方法具有对视网膜的经典组织学切片的几个主要优点：它可用于高通量筛选，并已被证明是一个有效的方法，用于鉴定调节PR存活和功能的因素。它可以在相同的生活ANIM用于PR变性的动力学分析人在几个星期内，向人们展示为在成年苍蝇公关生存或特定功能基因的要求。这种方法也用于解决在发育的突变体，如那些在其中建立的平面细胞极性影响细胞自主性的问题是有用的。

番茄/ GFP-FLP / FRT方法可用于研究的异位表达的突变或上的PRs在果蝇视网膜的发育和存活的影响。它是快速，非常适合筛查的目的，因为最近展示了20。突变体的PR的存在下一个要在同一个眼睛的野生型的PR可以很容易地检测与突变体的PR相关的缺陷，并解决在细胞自主性的问题。 在我们的分析中观察到的发育缺陷影响的各种流程，包括公关招聘，形态发生和平面细胞极性（PCP）的成立（ 图5）。 缺席七是必需的公关招聘，尤其是R7的内部公关之一。 七中在内部公关的损失和一些外永久居民（ 图5A） 缺席审判结果的损失。 木纹头（GRH）参与PCP建立和一些小眼是INVerted在GRH突变克隆（ 图5B）。这种方法可以识别马赛克小眼突变的PR，便于识别该基因是必需的正确采集五氯酚的PR。我们已经表明，GRH所需的正确采集的PCP在R3中前体20。的确，我们观察到，在倒置小眼，R4，它起源异常从R3前体，总是突变体。屑（CRBS）是必需的rhabdomere形态23的顶膜蛋白。 CRBS中在不规则的，更大或更小的PR（ 图5C）的CRBS 11A22突变效果的损失。 此方法也可用于跟踪单个的PRs成年期的命运（ 图6）。它是理想的神经退行性病变的研究，因为一组纯合​​突变的PR可以查看和同组可在的相同的眼睛来发现飞一样在几天内或几周。因此，可以判断哪些永久居民生存和它们注定会死，因为每个克隆具有独特的形状，当眼睛被定位在荧光显微镜下可予确认。如视网膜被极化，就可以以定向视网膜再次找到同一克隆，它的形状的基础上。我们用这种方法来证明FATP k10307突变诱导进步公关变性成年（ 图6,24）。这种可视化的方法也可以用于分析细胞的自主权。在FATP k10307马赛克视网膜，公关损失仅限于FATP突变永久居民，野生型的PR是不受影响。 FATP公关生存能力的规定是细胞自主。我们还可以通过reexpressing野生型FATP用RH1-Gal4的驱动器和一个UAS-FATP结构（ 图7），以拯救FATP突变公关。该进行这种救援实验的可能性是基于对树脂包埋的视网膜组织切片克隆分析番茄/ GFP-FLP / FRT方法的优点之一。的确，克隆的检测与番茄/ GFP-FLP / FRT方法并不受使用的P（UAS，W +）的转基因构建体，而与迷你白基因（W +）相关联的红色色素覆盖眼用红色颜料在组织切片掩蔽镶嵌克隆。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/50610/50610fig1.jpg" /> 图1。组织果蝇眼睛的。 （一）照片的果蝇眼睛用立体显微镜拍摄。 果蝇眼睛是由大约800小眼。的64小眼在眼睛中间的一个字段（B）示意图。这六个外光感受器的神经元每个小眼的（PRS），显示为绿色，被组织成一个梯形定型指向眼睛的距离最近的电线杆。因此，它们根据的两个部分，这是所谓的赤道之间的边界指向相反方向的腹侧和眼睛的背侧部分，并且镜像。一个小眼（C）的示意图。每个小眼包含八个永久居民。永久居民R1至R6，以绿色显示，有外永久居民。它们表达光敏分子rhodopsin1（Rh1的​​）并且是哺乳动物的视杆细胞的等价物。内的PR，R7和R8，以灰色显示（R7坐在R8的顶部）。内的PR表达视紫红质3，4，5或6，是哺乳动物的视锥细胞的等价物。为简单起见，只rhabdomere，公关的光敏感的部分，显示每个公关。 <a href="https://www.jove.com/files/ftp_upload/50610/50610fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <imgALT =“图2”FO：内容宽度=“4.25in”SRC =“/ files/ftp_upload/50610/50610fig2.jpg”/&gt; 图2。生成和可视化的马赛克公关克隆果蝇眼睛的番茄/ GFP-FLP / FRT方法。在番茄/ GFP-FLP / FRT方法镶嵌克隆的产生（一）示意图。通过有丝分裂重组产生马赛克的克隆，由于眼球发育和FRT序列在flipase（下无眼启动子的控制）的表达。后可以从杂合细胞会产生FRT序列和细胞分裂，纯合突变体，野生型纯合子和杂合子的细胞的重组。这些细胞再分裂形成永久居民的成年眼睛镶嵌克隆。突变的细胞的识别是通过将表达红色荧光蛋白tdTomato成野生型染色体，标记的野生型和杂合细胞的构建容易。马赛克PR克隆具有t（B-B&#39;）的可视化他番茄/ GFP-FLP / FRT方法。番茄/ GFP-FLP / FRT方法结合有丝分裂重组，角膜中和共聚焦显微镜。所有的PR表达绿色荧光蛋白，它便于使用角膜中和共聚焦显微镜（二）永久居民的可视化。收受可以在单细胞水平被观察到。通过有丝分裂重组产生的突变体克隆镶嵌，可以通过没有红色荧光蛋白tdTomato（B&#39;）的标识。因此，在合并后的图像，纯合子的PR显示为绿色，而野生型和杂合子的PR是黄色的（B&#39;&#39;）。 <a href="https://www.jove.com/files/ftp_upload/50610/50610fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/50610/50610fig3.jpg" /> 图3。设置果蝇的visualizat通过角膜中和离子。 果蝇的（A-A&#39;&#39;&#39;&#39;）照片固定在填充琼脂糖一盘。在A组&#39;&#39;&#39;&#39;，包含飞一块琼脂糖已被切断采取个人资料照片。 果蝇是半嵌在琼脂糖，用琼脂糖内的右翼和左翼贴在琼脂糖表面（A，A&#39;，A&#39;&#39;&#39;&#39;）。当果蝇嵌入在琼脂糖，其头部被定位常常不佳的眼睛（A，A&#39;）的可视化。头，因此必须重新定位，以镊子，使得眼睛的中间朝上（A&#39;&#39;，A&#39;&#39;&#39;）（B-B&#39;）表示果蝇示意图固定在琼脂糖板和的定位显微镜下的目标果蝇果蝇的设置。（C-C&#39;）上的舞台照片根据其目标显微镜。 <a href="https://www.jove.com/files/ftp_upload/50610/50610fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/50610/50610fig4.jpg" /> 图4。眼的时间过程分析的方向。图显示了一个果蝇头部在两个不同的方向和小眼的相应字段由角膜中和过的，表示为原理图的照片。（A）在第一次观察，映入眼帘的是放在这样的杂合或野生型的PR克隆（黄色）被认为在该领域的中间，在赤道上（红线）的水平。（b）于第二个观察，眼睛往往不完全相同相同的方向。因此，相同的组的PRs可以再次发现，但它不可能以查看完全相同的字段（左侧）。眼睛必须被重新定位为同一领域再次查看。赤道的位置可以作为一个指南。在这个例子中，我们知道，所观察到的字段是太腹侧和眼睛，因此必须转向腹侧放置在赤道中所观察到的字段的中间再次，如在第一观察。 <img alt="图5" fo:content-width="6in" src="/files/ftp_upload/50610/50610fig5.jpg" /> 图5。由番茄/ GFP-FLP / FRT法检测PR发育缺陷的例子。 （一）可视化的马赛克功略[BG02648]突变的PR，显示出内在的永久居民的损失。在突变小眼，外收受聚集在一起，由于缺乏内PR R7组成。事实上，新浪被称为是必需的内部公关招聘。马赛克GRH（二）可视化[S2140]突变的PR显示极性的缺陷。花叶突变体小眼，SURR由圆圈ounded，指向在从野生型小眼相反的方向，显示出背腹倒置。与番茄/ GFP-FLP / FRT方法的详细研究表明，GRH须在R3前体的正确采集小眼极性20。马赛克CRB [j1B5]突变的PR（C）可视化，显示出变形纯合子突变的PR。 CRB是已知的所需rhabdomere形态。 <img alt="图6" fo:content-width="6in" src="/files/ftp_upload/50610/50610fig6.jpg" /> 图6。马赛克FATP的时程分析[k10307]突变的PR与番茄/ GFP-FLP / FRT方法，超过14天。马赛克FATP [k10307]突变视网膜观察第1天（A，A&#39;），4日（ B，B&#39;），8日（C，C&#39;）和14天（D，D&#39;）孵化后。同组镶嵌永久居民的可FOUNð在每个时间点，在所观察到的场（白色矩形，A，B，C，D）。在永久居民（A&#39;，B&#39;，C&#39;，D&#39;）这个领域，纯合突变的PR被标记为绿色，而杂合和纯合野生型的PR被标记为黄色。从第4天（B&#39;）开始，纯合突变的PR开始消失，表明FATP [k10307]突变诱导这些减贫战略的逐步退化。 <a href="https://www.jove.com/files/ftp_upload/50610/50610fig6large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图7" fo:content-width="6in" src="/files/ftp_upload/50610/50610fig7.jpg" /> 图7。 FATP的救援[k10307]突变的PR马赛克控制的微型白色承载FATP表达构建。马赛克的PRS可视化，在15日龄果蝇番茄/ GFP-FLP / FRT方法（A）可视化永久居民。 （B）V马赛克FATP突变的PR，显示突变的PR损失马赛克（C）可视化isualization FATP在外部公关（RH1&gt; FATP）一只苍蝇reexpressing FATP突变的PR。突变的PR获救。迷你白色基因和相关的红眼色素沉着RH1的存在&gt; FATP条件并没有改变番茄或GFP​​荧光或克隆检测。

Watch this Scientific Journal Video about The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells at JoVE.com

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

番茄/ GFP-FLP / FRT方法涉及在活果蝇可视化马赛克感光细胞。它可以被用来追踪在视网膜上个别光感受器细胞命运数天或数周。这种方法是理想的视网膜变性和神经退行性疾病或光感受器细胞发育的研究。

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

the-tomatogfp-flpfrt-method-for-live-imaging-mosaic-adult-drosophila

Research

JoVE Journal

Biology

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

17.9K 次观看.  Ecole Normale Supérieure de Lyon. 番茄/ GFP-FLP / FRT方法涉及在活果蝇可视化马赛克感光细胞。它可以被用来追踪在视网膜上个别光感受器细胞命运数天或数周。这种方法是理想的视网膜变性和神经退行性疾病或光感受器细胞发育的研究。 

视频: The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

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 ...

科研

生物学

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

编制果蝇实时成像的胚胎使用悬滴协议

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

发展和成人的制备果蝇大脑和视网膜实时成像

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

电子自旋共振氧测绘活体物种显微成像

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

准备个人果蝇蛋钱伯斯实时成像

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...