The authors wish to thank T. Noguchi for helpful advice on the initial establishment of the culture technique in our laboratory. Research in the lab of R. R.-P. was funded by the DFG within the international collaborative research center TRR81.

道德声明： 在本协议中所述的实验过程涉及专门工作与果蝇和不受德国动物福利的法律。 1，准备媒体，工具​​和蝇<ol><li>制备改性市售奶粉M3增长率介质中无碳酸氢钾17。注意：刺激眼睛和皮肤。补充有10％胎牛血清的培养基中，100 U / ml青霉素和100微克/毫升链霉素。使用以达到最佳效果热灭活和昆虫文化测试的胎牛血清。过滤通过在等分0.2μm的瓶顶过滤器单元和存储区中的完全培养基（以下称为介质）在-20℃下。在使用之前，过滤器（0.2微米的注射器过滤嘴）的培养基中再次以除去沉淀物。 </li><li>准备抑制剂股票方案的药理试验。 Dissol已经在分装在适当的溶剂和储存的化学品。例如，准备28.69毫米的漆树酸原液：在二甲基亚砜（注意刺激眼睛和皮肤）。 </li><li>准备工具解剖蛹睾丸和单一囊肿剥离的破坏。用两个尖锐，坚硬的针头，而不是一对镊子。一对镊子的两臂之间产生毛细力，使单一的囊肿少量中非常困难的清扫。例如，使用不锈钢昆虫针插入接种环刀柄的顶端。 </li><li>为了获得适当的年龄蛹，种子约 10大培养瓶（管有直径为4厘米），与成年果蝇的足够数量的（ 约 40-60苍蝇）。分析高压开关，使用苍蝇表达H2AvD-RFP和ProtamineB-EGFP在其内生型12,16,22。培养在标准条件下将小瓶在25;℃进行约 4-5天，直至第三龄幼虫抓取并开始化蛹小瓶25的壁上。 </li></ol> 2，标记或收集白色预蛹获取24小时的老蛹解剖<ol><li>为了测试某些化学物质的高压开关上的影响，剖析蛹睾丸在约 24小时，薏仁12。要获取上演蛹，以准备飞文化小瓶脱蛹而出的幼虫（参见1.4节），并确定尽可能多的白色预蛹越好。期间在化蛹的第一步预蛹显示为静止的，非馈送，而缩短的幼虫与外翻气孔和白色puparium。该puparium变成褐色褐色于30到60分钟，这表明蛹发展26的下一个阶段。 </li><li>马克预蛹的位置上的小瓶的外部的永久标记。培养瓶中，在25℃下进行约 24小时。 <ol><li>另外，挑预蛹从小瓶的侧面用小刷子蘸用PBS缓冲液中。然后预蛹转移到新的50ml反应管的侧和孵育在25℃下进行约 24小时。 </li></ol></li></ol> 3，解剖蛹睾丸的Puparium形成后约 24小时<ol><li>分布在10厘米的塑料细胞培养物或培养皿中滴入几滴培养基（ 约每80微升）。使用广泛的钳子或沾介质画笔，挑蛹在从培养瓶中的24小时APF的阶段（见第2节）。传输一个蛹到中每滴上这道菜。 </li><li>对于夹层，使用立体显微镜配备了光纤照明器。解剖过程中，不要加热超过RT睾丸，因为这可能会妨碍正常发展的文化。 <ol><li>解剖蛹两对5号的镊子。有一对，抢蛹在最后部分（后气孔），并保持恰当的位置。 </li><li>抓住前气门与另一对钳子中，并在其前端打开蛹厣。剥离蛹壳，直到至​​少部分的胸部露出。 </li><li>继续通过其最后端保持在适当的位置蛹。要释放蛹的​​内部压力，将双臂一对钳入蛹的头部区域。 </li><li>瑞普蛹打开镊子和移动钳的前方向开。确保与该运动所产生的开口是尽可能的大，在第一次尝试。避免损坏蛹在其后端的压力已经释放之前，因为这可能会导致睾丸被直接推过小开口和最经常被打乱。 </li><li>注意，一旦蛹被打开，蛹的释放内部压力压出脂肪体细胞和组织的团块通过LARGE开口的头部区域。检查是否蛹睾丸已经推出了这种材料的蛹;这种情况发生在一些情况下。睾丸未连接到在24小时的APF任何其他组织，因此可能容易地从蛹23被排出。 </li><li>避免挤压蛹的镊子，因为这可能会损害睾丸，如果他们留在蛹。增加使用一对钳子的开口的大小。 </li><li>然后按住蛹在其最后端。关闭其他钳子，轻轻地走出连胜蛹的内容，从后到前，从蛹释放睾丸。 </li></ol></li><li>通过拉成一个预切加入200μl移液管尖端收集的睾丸。尝试传输只有极少量培养基，以减少与睾丸转移脂肪体细胞的数量。将睾丸进入中等新鲜培养皿。洗睾丸几次，直到中只有很少的脂肪体细胞S保持。 </li><li>对于实时成像，睾丸转移到玻璃底培养皿中，用一个倒置显微镜成像。药理试验，继续执行步骤5。 </li></ol> 4，解剖男性生殖系囊肿和实时成像的<ol><li>传送一个或多个蛹睾丸玻璃底培养皿。 </li><li>使用立体显微镜光纤照明和两个不锈钢针（见1.3），男性生殖系囊肿的剥离。 <ol><li>随着一针，小心翼翼地尽量拖住1睾丸在其前或后结束。保持恰当的位置。将其他针刺入睾丸，侧身移动针头破坏睾丸鞘膜。许多孢囊的将被从睾丸通过这种运动释放。 </li><li>继续保持在适当位置的睾丸有一个针。轻轻条纹从与其他针睾丸剩余的囊肿。 </li><li>单独汇总囊肿无论是绅士LY移动一针侧身通过囊肿的群集或用200微升枪头转移囊肿到一个新的培养皿中。 </li></ol></li><li>将培养皿以倒置显微镜成像单囊肿实时成像。用针在必要时再次分散囊肿。 <ol><li>通过使用相衬和荧光显微鉴定的包囊的阶段。 </li><li>专注于所期望的舞台囊肿。常常在较长的成像实验确认对焦和囊肿的位置，囊肿不坚持到培养皿的底部，因此往往会浮出来的重点。 注：涂层与聚-L-赖氨酸为固定的盘是不利的早期胚系囊肿的发展。相反，单独的囊肿可被分离。与一些实际应用中，也能够挑出特定囊肿由非常轻轻地用针头推动它。这将允许单一囊肿的转移到单独的崇拜尤尔菜肴巴斯德玻璃吸管一个漫长的提示，适合视显微镜领域。所述分离孢囊然后可以甚至在长期培养容易地重新定位在培养皿上。 </li></ol></li></ol> 5，药理学测定与完整蛹睾丸<ol><li>填充到24孔细胞培养板用500μl培养基的各孔中的一个实验中的12次重复。传输一个蛹睾丸，每孔，采用了预减200微升枪头。 <ol><li>检查精蛋白中分离的睾丸使用倒置荧光显微镜的存在。睾丸应该是免费ProtamineB-eGFP的信号，这表明该高压开关还没有发生任何的包囊的。更换睾丸已经表明ProtamineB-EGFP阳性囊肿。 </li><li>到第一的12个孔中，添加500μl的含有稀释的抑制剂化合物（漆树酸）介质中，以达到所需的最终浓度（150μM），在1毫升每孔培养基中。用剩下的水井未治疗的对照;加入500μl培养基用的溶剂用作与抑制剂化合物的量相同。注意：如果许多不同的化合物和溶剂的使用，这可能是更有效的，以单独使用介质作为对照，并测试在不同的实验中增加溶剂的浓度的影响。 </li></ol></li><li>孵育板在25℃下放置24小时，在黑暗中。 </li><li>再次检查是否有鱼精蛋白的存在下在温育睾丸如上所述。控制睾丸现在应该显示一个或多个ProtamineB-EGFP阳性囊肿，这表明通过HP开关过渡。 </li><li>用移液管以200μl的尖端，睾丸转移到聚-L-赖氨酸包被的载玻片，如别处12,27,28描述使壁球准备和染色用适当的荧光抗体。 </li></ol>

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作者没有利益冲突。

所提出的协议描述了解剖果蝇睾丸和单种系囊肿在给定的发育阶段以及对在培养皿的体外发展这些细胞和组织的能力基于两个两个不同的应用程序。一个应用是生命过程的精子发育过程中的药理操纵。重要的是，允许访问不容易使用基于动态遗传学分析功能得到减数分裂后精子。另一种应用是在显微镜下观察生活和发展中的生殖细胞和睾丸。特别是从果蝇细胞和器官的培养的能力本申请的好处生存，并在室温和常压下开发。因此，配备有加热阶段（加热到25℃，标准育种温度）倒置显微镜成像是足以获得在实时成像实验有意义的结果。更多此外，许多表达荧光蛋白标记的蛋白质的实时成像的转基因蝇菌株存在或可以容易地确定。 以获得特定的发育阶段的蛹睾丸或生殖系囊肿，至关重要的是，一个是熟悉果蝇发育的时序。每个阶段的确切时间可能实验室，培养条件而异，并且飞菌株，并且必须凭经验确定。如上文所述，这是为药理学试验，例如，针对高压开关尤其重要。对于这样的实验中，最好是总是检查精子与实际抑制剂试验前的ProtamineB-eGFP的信号，因为定时甚至可以在一个人口略有不同。 按照上述协议应使实验者解剖，从早期蛹的阶段睾丸免费的附加脂肪人体组织器官的完整。这些睾丸，更是这样，通用电气隔离RM行囊肿是很脆弱的。因此，关键的是要开发的手巧和必要的处理和使用镊子，针和移液管转移的睾丸和囊肿的经验。特别是针对减数分裂和早期减数分裂后的生殖细胞发育的研究将受益于这一点。虽然它是比较容易的解剖晚期精子细胞和成年睾丸长鞭毛囊肿，它是很难获得的早期阶段。解剖从下面这个协议的早期蛹睾丸囊肿这是更有效的。请记住，在一般的飞应变，蛹，只有约 50％是男性。因此，准备至少两次解剖许多蛹所需睾丸的数目。或者，也可以使用立体显微镜来识别男性L3幼虫，如睾丸可以识别嵌入在横向脂肪身体组织的完整幼虫23,34的半透明圆形器官，并收集它们在新鲜培养瓶中的可以用来接预蛹的分期和解剖。它也可能识别雄性预蛹35。 我们发现，无论是分离的生殖系囊肿和在培养的完整蛹睾丸对光敏感，如也已先前18报告。这光敏感度可能还保持在药理试验中使用的一些化学物质。因此，最好是保持在黑暗中的测定法的培养皿中孵育期间。同样地，规划时间推移成像实验开发睾丸，当特别的，孤立的囊肿，记住，过长的曝光时间和/或过高的采样率可能会抑制离体的发展。适当的采样速率应通过试验确定。 细菌和/或真菌感染和过度生长在培养皿上提出了严重的问题。感染培养皿中有太多的细菌不支持体外德韦讲习班中的睾丸和囊肿。因此，所描述的培养基中含有青霉素和链霉素（参见步骤1.1），但在长期的培养后，这些抗生素可能变差和它们的活性可能降低。这可以是一个使用上述的协议时，观察到培养的囊肿的原因的生存时间有限（最多72小时）。然而这个时间帧不能被定期更换的菜肴培养基中进行扩展。我们得出结论，其它因素可能影响生存中培养，如缺乏由生殖道的其它组织生长信号。另一方面，减数分裂后发展是在果蝇快于哺乳动物。在果蝇中 ，精子需要大约90小时，和HP交换机将减数分裂9,12后50至60小时之间。因此，围绕48小时取得与该协议通常的存活时间是非常适合于跟随在SPE关键发育过程rmatogenesis，如减数分裂或HP交换机。虽然细菌或霉菌污染，可避免使用清洁工具和媒体所提出的协议不利用严格的无菌条件下工作，因为苍蝇和飞睾丸已经包含细菌时，在标准条件下提出的。然而，这种文化技术的一些应用程序可能会受益于解剖苍蝇，甚至在无菌条件下饲养（对协议 ​​，见17,36,37）。 药理试验依赖于使用作为抑制剂或多种酶的活化剂的化学化合物。在这样的试验中常用的化合物，常常有很大的不同在他们的靶特异性。作为一个优点，这提供了针对整个酶类别的电位，例如，用漆树酸抑制P300 / CBP，PCAF，和帽子38的MYST家族成员。这种方法的缺点可能是潜在的脱靶或细胞毒性作用INDUC这类化合物主编。因此，与蛹睾丸或囊肿的测定法中使用的每种化合物应该为它的毒性和不同浓度的特定效果进行测试。此外，在培养条件下，化合物的浓度或活性的和变化中的细胞渗透性的微小变化可能导致的诱导的作用的变异性。因此，有必要监测在每个实验中的治疗的成功。例如，当我们用漆树酸，在150μM，我们观察到轻微变异之间，有时在某些睾丸的染色质缩合型的强度，而组蛋白H4的乙酰化持续减少。 药理学试验与果蝇睾丸中培养已被用于分析精子4,12,14,21的预处理和后减数分裂的机制。因为它是难以进入减数分裂后精子与遗传工具针对玩家essentia升预减数分裂和减数分裂的功能，这种体外方法构成了一种替代方法。此外，原则上，该方法可以适用于脉冲追踪实验遵循治疗生殖细胞的发育时间的推移。然而，我们还没有测试过这种可能性。利用初蛹睾丸是在药理学试验的优点。首先，年轻的蛹睾丸（约24小时，APF）的薄外套可以让化学物质的提高透气性。其次，在研究后减数分裂高压开关，蛹睾丸的解剖与鱼精蛋白表达GFP的飞线24小时薏仁能的高压开关是否受影响或不直接读出。 迄今为止，已经开发了用于体外培养的果蝇的器官和组织，包括睾丸和生殖系囊肿多个协议（ 例如，见4,14,17,20,39,40）。栽培介质和使用的一般条件在这里介绍的是成立于1979年的协议17。培养体系，后来适应了体内成像的个性化机制，在后期减数分裂后囊肿成人睾丸4隔离。此前，我们曾报道，这些条件也支持生存和减数分裂和早期减数分裂后的生殖细胞分化的囊肿约48小时12。相对于其他文化系统19在这些文化所观察到的发展时机与固定样本确定（有关详细信息，请参阅第12）的时间基本一致。 H2AvD-RFP和鱼精蛋白-GFP荧光信号可用于可视化的生殖细胞中培养的核形态和染色质的组合物。我们发现，对于明确识别在培养发育阶段，这是有利的，在其它条件，如卷取囊肿。重要的是，所提出的协议不涉及除飞转的RACT或其它生长因子比胎牛血清等。此外，我们在这里表明，该条件与减数分裂离体培养的荧光显微成像相容。影像可以在支持长期发展一个易于使用的媒体合理的解决方案来获得。这是相对于协议实现短期（ 约 3小时），观测在Voltalef油41,42。 上述用于种植和药物治疗蛹睾丸和单身男性生殖系囊肿的程序是很容易适应的许多基因，表观遗传学，细胞生物学，或发育问题，可以在果蝇的精子进行研究。这特别包括研究着重于生殖系干细胞。它已经表明，干细胞的发育，可接着培养的成年的实时成像睾丸20,43。然而，在成熟睾丸的蠕动运动sheatħ干扰图像采集。因此，无论是成像是使用零散睾丸43或进行成像实验的数量必须增加以考虑丢失的数据集20。蛹早期睾丸（24小时APF）尚未封闭的肌肉细胞。即使是年龄稍大的睾丸（36-45小时APF）的出现，显示几个蠕动（对比图3和补充视频1）。因此，培养年轻的蛹睾丸作为完整的器官可以作为一种替代方法。 此外，一旦掌握，解剖蛹睾丸的能力，并最终分离生殖系囊肿将允许使用单个孢囊作为一种均匀的来源，虽然小，细胞群的进一步应用。例如，它因此能够进行RT-qPCR的分析特定基因的过程中精子的限定阶段的转录调控，如已经证实<suP&gt; 15。

许多物种的雄性生殖细胞的发育是一个连续的过程。它的特点是一系列关键事件的高潮在形态和表观遗传学专业性强精子的形成。在果蝇 ，种系干细胞在睾丸鸿沟尖不对称，以产生新的干细胞和精原细胞，也就是说 ，致力于分化的子代细胞（ 见图1概述）1,2 。精原细胞经过四个回合有丝分裂和减数分裂用的发作，令人印象深刻的增长和转录活性的阶段叫做精母细胞阶段。将细胞从一个精原细胞始发保持连接到彼此和开发作为同步包裹由两个体细胞 - 一种结构称为囊肿。减数分裂完成后，将雄性生殖细胞的形态是完全重组（示意于图1中示出）。最初，圆精子细胞伸长，以形成流体动压头结构，以及鞭毛发展。 5 -最终，精子细胞彼此通过所谓个性3的复合物，细胞凋亡相关机制分离。 在许多种，包括果蝇和哺乳动物物种的生殖细胞都伴随着单倍体雄性生殖系染色质的一个显着的表观遗传重排的形态的变化，被称为组蛋白对鱼精蛋白开关（HP开关）6 - 9。可能几乎所有规范组蛋白和许多组蛋白变体分子从DNA的剥离和由小的碱性蛋白质，在鱼精蛋白，从而导致特定于成熟精子染色质的高度紧凑的精蛋白的结构所取代。惠普交换机的一般特性为t他替换规范组蛋白的第一个由组蛋白变体，然后通过过渡蛋白，最后通过鱼精蛋白。所有这一切都伴随着一些改变表观遗传标记，如组蛋白H4的乙酰化水平激增之前，为了去除组蛋白7,10 - 12。 大多数，如果不是全部，上述过程是成熟的，完全可育的精子的发展至关重要。这不仅是在果蝇中，而且在人类中为真，如哺乳动物精子股与无脊椎动物系统1,8,9相当量的相似性。研究男性生殖细胞发展极大地促进了果蝇模型系统。果蝇在遗传上非常方便。突变的产生，以及建立即时株表达融合基因或RNA干扰结构需要多大的努力。然而，在减数分裂后精子发生，利用突变体的研究中ð简单的遗传工具可能会达到一个极限。在果蝇精子发生，转录停止几乎完全进入减数分裂。因此，减数分裂后精子的主要依据是在扩展减数分裂前期13合成翻译压抑和存储的mRNA - 16。因此，如RNA干扰或使用非条件突变体工具不适合用于学习具体的基因产物也满足在预减数分裂或减数分裂的生殖细胞发育所必需的功能的减数分裂后的角色。 果蝇的一个优点是，可以在这样的情况下被利用是解剖完好睾丸和生殖细胞甚至囊肿开发体外培养液中4,12,17的能力- 20。睾丸或生殖系孢囊的解剖和培养可以在活细胞中不仅显微镜观察生殖细胞的发展，但LSO使用药理试验用中， 例如：酶复合物的抑制剂，例如组蛋白乙酰转移酶（HATS），组蛋白脱乙酰酶（HDACs），拓扑异构酶，或蛋白酶。由此，能够减数分裂后精子发生过程中操纵酶功能和监视的发展结果不论胚胎，预减数分裂或减数分裂功能4,12。 在药理试验，我们以前分析了布罗莫蛋白在减数分裂前期的乙酰化相关的本地化版本，以及组蛋白乙酰化水平在减数分裂后精子的后生惠普交换机通过使用针对帽子和12,21的HDAC抑制剂具体的相关性。针对这些试验的高压开关成为可能通过使用飞菌株表达的融合基因histone2AvD-RFP和protamineB-EGFP 12,22的内生模式，通过观察发现蛹睾丸的精子首先经过24及48小时薏仁12之间的高压开关。 提出的协议描述了果蝇蛹，培养条件和药理实验与完整蛹睾丸和睾丸囊肿的剥离。为了这个目的，在puparium形成（APF）后约24小时，蛹睾丸解剖（参见图1和图2）。此时，睾丸没有作连接至其它组织和仅由薄的外皮，这允许更好地渗透通过该抑制剂化合物23,24包围。睾丸是可以很容易地考虑到培养bean-或梨形的器官。这些睾丸解剖，进行培养，并用特异性抑制剂治疗。接着，将抑制剂的效果是使用免疫荧光分析，并融合蛋白表达的荧光显微镜监测，并通过核morphol的比较术处理过的细菌细胞，以未处理的生殖细胞中。在这个协议中提出的条件，也允许开发睾丸和生殖系包囊在培养的实时成像。

<table border="0" cellpadding="0" cellspacing="0" width="782">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Anacardic acid</td>
			<td style="width:105px;">Merck Millipore</td>
			<td style="width:99px;">172050</td>
			<td style="width:247px;">brand: Calbiochem (EMD Millipore)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-acetyl-Histone H4</td>
			<td>Merck Millipore</td>
			<td>06-598</td>
			<td>brand: Upstate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AxioCam MRm&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AxioObserver.Z1 inverted microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont 5-Inox forceps</td>
			<td>Dumont</td>
			<td></td>
			<td>multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum, Heat Inactivated</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fiber optical&#160; illuminator</td>
			<td></td>
			<td></td>
			<td>multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Bottom Microwell Dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">goat anti-rabbit IgG (H+L)-Cy5</td>
			<td>Dianova</td>
			<td>711-175-152</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoechst</td>
			<td>Sigma</td>
			<td>B1155</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Inoculation loop or pin holder</td>
			<td></td>
			<td></td>
			<td>multiple suppliers</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Austerlitz INSECT PINS stainless steel needles, size 0</td>
			<td>Plano GmbH</td>
			<td>N5018</td>
			<td rowspan="2" style="width:247px;">In our hands both these pin sizes worked well in the dissections. The minutiens are less rigid but have a smaller tip diameter.</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">Austerlitz INSECT PINS stainless steel minutiens, diam. 0.1 mm</td>
			<td>Plano GmbH</td>
			<td>N5014</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multiwell plates, 24-wells</td>
			<td>Becton Dickinson</td>
			<td>351147</td>
			<td>brand: Falcon</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pen Strep</td>
			<td>invitrogen</td>
			<td>15070</td>
			<td>brand: Gibco</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Shields and Sang M3 Insect Medium</td>
			<td>Sigma</td>
			<td>S8398</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stemi SV6 binocular</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo culture of drosophila pupal testis and single male germ-line cysts: dissection, imaging, and pharmacological treatment

在精子发生在哺乳动物和果蝇，雄性生殖细胞发育了一系列重要的发育过程。这包括从干细胞群，有丝分裂扩增，和减数分裂分化。此外，减数分裂后的生殖细胞经受急剧形态重塑过程以及生殖系染色质组蛋白对鱼精蛋白交换机的全局后生重新配置。 研究一种蛋白在减数分裂后精子通过诱变或其他遗传工具的作用往往是由必要的胚胎，预减数分裂，或者正在调查该蛋​​白的减数分裂功能受阻。这样的蛋白质的突变体的减数分裂后的表型可以通过较早的发育块被遮蔽，或表型的解释可能是复杂的。模式生物果蝇提供了一个绕过这个问题：完整的睾丸和从早期的蛹解剖生殖细胞囊肿甚至能够开发体外培养液中。利用这种文化使得在种系囊肿和睾丸生精活细胞显微成像。重要的是，培养睾丸和生殖细胞也成为访问药理抑制剂，从而使操纵酶功能的精子发生过程，包括减数分裂后阶段。 提出的协议描述了如何剖析和培育蛹睾丸和生殖系囊肿。在蛹的睾丸和培养条件的发展信息，提供现场旁睾丸和生殖系囊肿文化显微镜成像数据。我们还描述药理实验研究减数分裂后精子，通过针对使用组蛋白乙酰转移酶抑制剂漆树酸的组蛋白对鱼精蛋白开关的实验例证。原则上，本栽培方法可以适用于处理许多ö疗法研究问题的前，后减数分裂精子。

已经形成在果蝇雄性的睾丸中的胚胎，并在幼虫阶段仍然存在于体腔。第三龄幼虫显示小的圆形的睾丸被一层未来色素细胞包围和嵌入在脂肪身体组织（ 图2A）。成年雄性果蝇精巢长，盘管，这是覆盖了一层肌肉细胞从生殖盘和颜料层24发起的。有趣的是，幼虫睾丸只包含生殖系前减数分裂和减数分裂期的细胞，直到初级精母细胞阶段，其对应于减数分裂前期（也参见图1）23。雄性生殖细胞的第一队列中puparium形成通过减数分裂。在约 24小时的APF，具有细长的鞭毛减数分裂后阶段已经开发，以及所有的细胞核含有组蛋白H2AvD-RFP（ 图1和图2B）。概无精子的经历吨他的HP交换机作为无ProtamineB-eGFP的信号可以通过荧光显微镜（ 图2C）来检测。频繁，解剖在此阶段睾丸含有可变大小让人联想脂肪体细胞的团块或1的一个自动荧光结构几滴油状物（ 图2C中 ，箭头）。睾丸内该结构也与相衬显微镜可见的。到今天为止，我们还没有发现它在文学性质的描述。睾丸解剖在36小时APF看起来拉长并且在某些情况下，已经开始卷曲（ 图2D）。他们已经附着在生殖道从生殖器成虫盘23,24生长出的组织。此外，它们经常含有超出高压开关的第一精子细胞，由ProtamineB-eGFP的信号（ 图1和图2D所示 ，箭头）所指示的。在48小时的薏仁，睾丸有进一步拉长，并明确在开始卷发近端。几种系孢囊与鱼精蛋白阳性细胞核变得可见（ 图2E，箭头）。 当睾丸 ​​解剖在24小时APF保持在培养24小时，它们不细长作为完整蝇（比较图2D和2E与图6A&#39;）。这可能是由于缺乏通常从显影生殖道在其后侧端部23,29接触的睾丸发送位置和生长信号。另外，睾丸鞘膜的肌肉层不显影因为新生肌管不能从生殖器圆盘迁移到睾丸24。睾丸鞘在这种情况下只薄的颜料层似乎并没有充分生长在文化信封越来越多的发展中国家在生殖细胞。然而，生殖细胞生长，分化，独立于睾丸鞘膜的区别。因此，经过36小时R IN文化，早期的睾丸经常会爆开，再发展没有观察到。然而，在较短的时间内，它可以记录的时间推移的体外活整个睾丸中培养显影的图像，如由图3A -3℃（参见补充视频1）。蛹期睾丸，解剖在约 45小时薏仁，培养在培养基中6小时，拍摄每5分钟。在这段时间内，精子数囊肿从HP交换机舞台崭露头角，并显示一个明亮的ProtamineB-EGFP信号（ 图3A&#39;-3℃，空心箭头）。 如上面所提到的，雄性生殖细胞在果蝇睾丸开发作为互连单元的同步包，命名囊肿1。 图4A示囊肿从蛹睾丸解剖在约24小时的APF的概览。前减数分裂期，减数分裂阶段，早期减数分裂后阶段，也囊肿在早期独木舟阶段之前与惠普开关细长晶核可以发现（ 图1和图4B -4 E）。孤立囊肿是很脆弱的，必须小心处理。形成的囊肿的包络在两个体细胞可以容易地破碎机械。生殖系干细胞具有生存和继续的体囊肿细胞的遗传消融后除以体内 30的能力。已经报道了在体外实验中，16个细胞期的生殖细胞从它们的体壳分离进一步发展和分化19。事实上，我们注意到，在我们的推测后期精母细胞完成减数分裂与破坏囊肿细胞，虽然很罕见的文化体系。最常见的，这些破坏囊肿停止发展。以下所提出的协议，完好囊肿可能在某些情况下，dev的埃洛普体外培养基中最多72小时的。但是，我们观察到囊肿合理数量的存活长达48小时的潜伏期。在这个时间帧，将培养的孢囊是能够从初级精母细胞阶段到发展直到减数分裂后，以及从圆形核阶段与组蛋白为基础的染色质，直到HP以外已故独木舟级开关12。这允许在精子发生生命过程的实时成像，例如，精母细胞进入减数分裂（ 图5和参考视频2）。对于这个实验中，RFP标记的组蛋白变体H2AvD用作染色体标记物。初级精母细胞阶段约 3 天 ， 体内 31延伸。因此，为了记录减数分裂，我们选择孢囊与已明显开始染色质凝聚（ 图5A，区域2）精母细胞。减数分裂开始在精母细胞对囊肿的一侧，然后如下在其余精母波（ 图5A中从区域2到区域1中，也见参考视频2）。的染色质缩合很快导致快速移动的染色体是作为亮点（ 图5A，区域2）中可见。 30分钟后，此区域的第一精母细胞已经处于末期（ 图5B中 ，箭头）。年轻的区域1的染色体排列在中期板20分钟后（ 图5C）。他们完成末期，并准备约15分钟后（ 图5D）经过细胞分裂。后期，末期和胞质分裂一直持续在该记录（ 补充视频2） 约 10分钟。 在哺乳动物中，以及在果蝇中组蛋白H4乙酰化的显着增长在减数分裂后，标志着去除组蛋白和HP交换机发作精子6,11。已经提出，这种表观遗传修饰是必需成分，甚至触发惠普的发育程序的开关10,32。以前，我们处理果蝇蛹睾丸文化与抑制剂靶向帽子和的HDAC在减数分裂后阶段12来影响组蛋白H4的乙酰化水平。在这些药理试验中，我们发现，组蛋白乙酰化是对精子发生的从与组蛋白为基础的染色质与鱼精蛋白为基础的染色质级阶段的进展至关重要。 图6和图7示出了使用同时含有漆树酸靶向蛹睾丸教的药理试验的代表性结果。睾丸在24小时薏仁从表达H2AvD-RFP和ProtamineB-EGFP苍蝇株进行了解剖。 ProtamineB-EGFP蛋白中不存在这些早期蛹睾丸（ 图6A和B）。经过24小时R中培养，所述控制睾丸表明ProtamineB-eGFP的信号，这表明在第一包囊已经发展超越惠普开关（ 图6A&#39;，开放箭头）。这是不与150μM漆树酸（ 图6B&#39;）处理睾丸的情况。睾丸壁球制剂和免疫荧光分析，提供更详细的信息（ 图7）。在未处理的对照，在比较大的初级精母细胞的细胞核可以通过其特征的丰富染色的DNA的三个区域，它们分别对应于主要的染色体果蝇 （ 图7A中 ，第1列）的（4C级）模式进行识别。组蛋白H4的乙酰化是清楚地检测到在这一阶段（ 图7B中 ，第1列）。减数分裂后的第一级被称为Nebenkern阶段，其特征是相当大的，圆形核（参见图4C，在图7中未示出）。在图7所示的下一阶段是由鞭毛生长和生殖细胞的细胞核，这已经比在较早的阶段（ 图7A，第2列）小得多，显示出非常低的组蛋白H4的乙酰化几乎检测不到的水平的圆形状识别（ 图7B，第2栏）。圆形状，然后拉长（ 图7A，第3栏），以及组蛋白H4的乙酰化是再次清楚地检测到（ 图7B，第3栏）。在精子细胞，然后到达独木舟造型阶段，其中高压开关发生和组蛋白被鱼精蛋白取代（ 图7A - 7℃，列4和5）。以下的独木舟阶段，protaminated晶核然后进一步拉长成非常细的针的形状，在成熟精子（此处未示出）。 用漆树酸处理过的睾丸睾丸壁球准备表现出不同的结果（ 图7D &lt;/ STRONG&gt; - 7 F）。在用漆树酸处理过的细菌细胞中的染色质比未经处理的对照（比较图7D，处理和图7A，控制）的出现更稠。免疫荧光分析表明，H4的乙酰化被大大削弱，甚至在漆树酸（ 图7E）治疗生殖细胞的细胞核缺席。已知的是高度乙酰化的组蛋白与开放的染色质区域相关联，而缺乏组蛋白乙酰化的染色质区域经常显示出压迫性结构33。因此，它是不令人惊讶的是与漆树酸处理的影响的处理的生殖细胞的细胞核的染色质结构。重要的是，独木舟后期阶段或以后没有鱼精蛋白阳性细胞核被确定在治疗睾丸（ 图7F）。因此，我们的数据是一致的想法是一致的HAT活性是必不可少的精子进行来回米蛋白为基础的染色鱼精蛋白为基础的染色质的阶段。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51868/51868fig1highres.jpg" width="500" /> 果蝇精子发生的图1概述。左侧面板显示阶段的生精细胞发育的干细胞，以个性化的精子序列。示意图显示了生殖细胞的特征核形态。一个精原细胞分裂，产生16互联的精母细胞在一个囊肿。在此阶段所需的大部分减数分裂后发展的转录物的产生，翻译抑制，并进行存储。与进入减数分裂的转录活性的堆积结束。组蛋白对鱼精蛋白在染色质开关需要早期和晚期独木舟阶段之间发生。阶段与组蛋白为主的染色质高亮红色;用鱼精蛋白为基础的染色阶段以绿色突出显示。在右侧面板中的横道生殖细胞，可以在正常的开发睾丸中找到幼虫，蛹在约 24级和36小时puparium形成后（APF），和成蝇。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51868/51868fig2highres.jpg" width="500" /> 图2 果蝇精巢处于不同的发展阶段。 （一）第三龄幼虫（L3）的稍压扁全安装睾丸相衬图像。只有前减数分裂和减数分裂阶段都可以找到。精原细胞被限制在毂区域（星号）的下方前部尖端。剩余的睾丸充满精（空心箭头）（B）稍微压扁睾丸在约的相衬图像<html> puparium形成（APF）后24小时。除了 ​​精母细胞（空心箭头），睾丸中含有的Nebenkern级精子（开放箭头），圆核和精子细胞的延长阶段，不断增长的鞭毛（双开箭头）（C - E）整个安装蛹睾丸表达H2AvD-RFP和ProtamineB-EGFP（的相衬和EGFP和RFP的荧光图像叠加）（C）蛹睾丸解剖在约 24小时的APF。箭头标志着自体荧光推测脂肪体细胞。 四）蛹睾丸解剖在约 36小时薏仁显示第ProtamineB-EGFP阳性囊肿（箭头）（E）蛹睾丸在约 48小时的APF与一组ProtamineB-EGFP-阳性囊肿（箭头）。在所有图中零件，星号表示在轮毂的区域。比例尺：100微米。ANK“&gt;请点击这里查看该图的放大版本。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51868/51868fig3highres.jpg" width="500" /> 图3 Live中的组蛋白对鱼精蛋白开关成像在蛹睾丸越来越体外 。 （一）蛹期睾丸表达H2AvD-RFP和ProtamineB-EGFP在约 45小时puparium形成（APF）后，考虑到文化的相衬图像。精囊是由实心箭头表示。 àparagonium（双箭头）仍附着于睾丸（A&#39;）中的（A）所示的睾丸EGFP和RFP的荧光。开箭头表示ProtamineB-EGFP阳性囊肿。比例尺：100微米（B）2小时30分钟，在文化，一些进一步的囊肿（空心箭头）后出现FR嗡组蛋白对鱼精蛋白过渡（C）之后的附加 ​​长约3小时，在文化， 即共培养5小时29分，一组囊肿（空心箭头）具有较强的ProtamineB-EGFP信号是睾丸的近端可见。另见补充视频1。在所有图件，星号表示该枢纽地区。 <a href="https://www.jove.com/files/ftp_upload/51868/51868fig3highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51868/51868fig4highres.jpg" width="500" /> 图生殖细胞的4隔离囊肿处于不同的发展阶段。 （一）囊肿从睾丸解剖在puparium形成（APF）后约 24小时。叠加的相衬和H2AvD-RFP的荧光（红色）的图像。比例尺：100μm左右。（B - C）单一囊肿不同阶段的放大倍率。叠加的微分干涉对比（DIC）和H2AvD-RFP的荧光图像。比例尺：20微米（二）16精母细胞囊肿64圆形精子细胞（C）的囊肿在Nebenkern阶段。该Nebenkern结构从稠线粒体开发并在DIC图像上可见的旁核（开放箭头）。精子与轮H2AvD-RFP-阳性细胞核并拉伸鞭毛（D）的孢囊（开放箭头表示伸长Nebenkern结构，伴随着不断增长的轴丝）（E）精子的囊肿在早期阶段的独木舟出一个细长的核形的根尖顶端。在广泛细长的鞭毛只是部分可见。 <a href="https://www.jove.com/files/ftp_upload/51868/51868fig4highres.jpg" target="_blank">请点击这里查看该F的放大版本。</a>igure。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51868/51868fig5highres.jpg" width="500" /> 一个16精母细胞囊肿发生囊肿的文化减数分裂I 体外 。荧光图像表达H2AvD-RFP 的图5实时成像 。描绘的是减数分裂一，精母细胞中的囊肿通过在波浪状的方式减数分裂特性阶段：（A）染色体缩合开始在细胞核的囊肿（2）的一侧，并前进通过囊肿（方向由箭头指示）。细胞核中的相对的区域（1）代表的染色质缩合，其中组蛋白密集区域标记的三大染色体仍然可以辨别的早期阶段。在区域精母细胞（2）含有完全冷凝染色体，可见明亮的荧光斑点。（二）30后0;分钟，在区域中的第一精母细胞（2）是在末期（开放箭头），而染色体缩合继续在区域（1）（C）在区域中的最后一个精母细胞（1），染色体（箭头）排列。在中期板20分钟后，（D）在另15分钟，最后精母细胞似乎已完成末期和现在准备进行胞质分裂（开放箭头）。另见补充视频2比例尺：50微米<a href="https://www.jove.com/files/ftp_upload/51868/51868fig5highres.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51868/51868fig6highres.jpg" width="500" /> 图6：漆树酸抑制组蛋白对鱼精蛋白在培养蛹睾丸开关。蛹睾丸expressi<html>纳克H2AvD-RFP和ProtamineB-EGFP和puparium形成后解剖，在24小时（APF）孵育24小时，在培养基中不含抑制剂（对照A，A&#39;，DMSO，二甲亚砜），或在培养基中添加150μM的漆树酸（ B，B&#39;）。用星号标明毂区域（A，B）没有ProtamineB-EGFP-阳性囊肿可剥离后进行观察。（A&#39;）后，培养24小时后，一些囊肿行的组蛋白对鱼精蛋白开关和ProtamineB-EGFP阳性用漆树酸处理过的孢囊（开放箭头）可以观察到（B&#39;）的睾丸不发展ProtamineB-EGFP-阳性囊肿。比例尺：100微米<a href="https://www.jove.com/files/ftp_upload/51868/51868fig6highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> 68 / 51868fig7highres.jpg“WIDTH =”500“/&gt; 图7漆树酸会影响组蛋白H4乙酰化和精子细胞的染色质结构 ，从蛹睾丸精子细胞的细胞核（24小时APF）的表达ProtamineB-EGFP培养24小时无抑制剂（A - C）的壁球准备。或150微米漆树酸（D - F）。 DNA用Hoechst染料（A和D）。 H4的乙酰化免疫荧光分析（B和E）。存在鱼精蛋白与ProtamineB-EGFP信号（C和F）监测的。以漆树酸处理后，将染色质出现强烈压实（D）和H4的乙酰化信号中所有精子细胞阶段（E）完全还原。此外，漆树酸处理过的睾丸显示独木舟后期阶段，或超出无囊肿，没有ProtamineB-EGFP信号（F）。比例尺：10微米<a href="https://www.jove.com/files/ftp_upload/51868/51868fig7highres.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> 补充视频1，通过绿色荧光蛋白和组蛋白对鱼精蛋白开关的RFP荧光在线成像在蛹睾丸生长 6小时的时间过程的成像体外 。视频示出了通过增加ProtamineB-的患病组蛋白对鱼精蛋白过渡绿色荧光蛋白阳性囊肿。图像获取的，每5分钟。有关详细信息，请参阅图3。（请参阅“suppl_video_1.MOV”下载下）。 一个16精囊肿发生减数分裂补充视频2在线影像í 体外 。一时间推移荧光成像囊肿文化表达H2AvD-RFP超过80分钟的过程。这部影片描述了一减数分裂图像的特征阶段被收购每隔1分钟。有关详细信息，请参阅图5。（请参阅“suppl_video_2.MOV”下载下）。

Watch this Scientific Journal Video about Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment at JoVE.com

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

解剖，成像和药物治疗： 果蝇蛹睾丸和单身男性生殖系囊肿的体外培养

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

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Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

17.3K 次观看.  Philipps-Universität Marburg. This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

视频: Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

果蝇幼虫NMJ夹层

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

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 ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

前初级人类输卵管上皮细胞的体外培养

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

果蝇蛹腹部免疫

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

通过影像学的蛹壳<em&gt;果蝇</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

小鼠胚胎皮肤和Live成像黑素细胞迁移的体外培养

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

在小鼠睾丸活体显微注射和电穿孔

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

解剖，成像和药物治疗： 果蝇蛹睾丸和单身男性生殖系囊肿的体外培养

解剖，成像和药物治疗：果蝇蛹睾丸和单身男性生殖系囊肿的体外培养