作者感谢NSF（MCB-1157876）提供资金。我们也感谢阿尔贝托CIVETTA约翰Roote，两位匿名审稿征求他们的意见。

应进行小规模的实验，熟悉整个过程。 1。 ，收集处女女性和朴素男性概述实验的最简单的版本包括四种类型的初始十字架，其中涉及下面结合成人：）W 1118人，以便收集处女雌二）俄勒冈-R，以收集天真参考男性的个人; ）P {SW}纯合子雄性和雌性携带野生型组织为了收集的天真实验男性（I型）， 国投的控制线;和d）P {SW}纯合子的男性和国投SW缺失携带处女雌为了收集天真实验国投SW缺乏（II型）的男性进行。 <ol><li>设置多个小瓶含8-10名女性和5名男性。允许女性奠定鸡蛋和新的小瓶传输成人每隔3-5天。使用瓶，而不是必要的小瓶，如果总是用新鲜的食物。所需的小瓶的数量将依赖于所研究的实验男性类型的数目，估计是要检测的差异（ 表1）的数目的个人。每瓶10多名女性，可能会导致过度拥挤在幼虫生长，这可能会导致在生育后代的变化。商店瓶在25°C的温度控制室。 </li><li>开始收集处女女性和天真男性的上11 日和12 日成立后最初的十字架。等待的时间根据不同的温度较低的温度下在较长的发展时间延迟个人的集合。羽化的影响的定时的另一个因素是不同的介质。营养丰富的食物，如玉米面酵母培养基29的成人，确保正确的发展生殖系统，这有利于配合。收集未交配的苍蝇，每4-6小时。当收集，引入瓶中的CO 2，麻醉苍蝇点击苍蝇，性别体视显微镜下（ 图2）。按性别和表型和适当标签的小瓶，将所需的苍蝇成不同的小瓶。 </li></ol>注意事项1。收集日常在早上开始丢弃前一天晚上出现在成人。收集处女女性和天真男性的一次或两次在白天进行。通常情况下，D。成为性成熟的雄性果蝇羽化后的8小时，在25°C 30。如果的苍蝇都保持12:12小时的光/暗周期下，预计两峰羽化：在最初的1-2小时后灯开启，并在2个小时前的指示灯会熄灭。羽化后24小时内发生的蛹变暗。 注2。女性不超过10 SH应投入相同的小瓶，以防止过度拥挤。这也限制了损失的女性的情况下，他们必须被丢弃，因为它们中的至少一个不是处女。 注3。为了收取适当数量的的处女女性和男性天真在很短的时间内，可以采用以下措施。设置实验中，涉及两种类型的实验男性（ 表1）1118 瓦特 15-20小瓶。轻轻地洒在介质表面，并添加一些活性干酵母颗粒，以方便产卵。传输父母至少4次，连续几天。化蛹在每个管形瓶中，以增加可用的表面，插入多折叠纸（7×5厘米），在第 4或第 5天后，父母被转移到另一小瓶中（ 图3）。如果羽化数从最初的十字架是不够的，等再过几天，收集个体ALS从小瓶设立在不同的日期。计划开展的实验，甚至连续几天的工作量。 2。双交配实验 图4示出了双交配实验14中所涉及的主要步骤。 <ol><li> 1日上午，成立了第一个交配。俄勒冈-R的雄性交配的罪行检测。 <ol><li>使用吸引器，设立小瓶含10 ,4-5日龄白眼1118瓦特处女的女性和10红眼俄勒冈-R天真男性。连续5天，每天两到三瓶。要设立的瓶数可能会有所不同，这取决于可用的个人的数目。允许苍蝇交配2小时。 </li><li>丢弃俄勒冈-R男性和放置到一个新的小瓶，使用吸气器（ 图5）的每一位女性。性别鉴定可以通过目视检查几个形态上的差异（ 图2）。适当地标示每个小瓶，离开女性在小瓶为2天（以下简称“卷”）。 </li></ol></li><li>第3天，成立了第二次交配。男性和交叉设置应进行随机选择，为了尽量减少任何潜在的偏向任何实验的男性类型。 <ol><li>前两小时指示灯会熄灭，再次转移到一个新的小瓶女性吸气。适当地标签新瓶（以下简称“V2”）。 </li><li>介绍5-6日龄三个实验男性相同基因型到V2。 </li><li>重复2.2.1和2.2.2为每个女性。 </li><li>第4天，2小时前光开启（ 即不超过12个小时后，2.2.1），丢弃男性使用抽吸。 </li></ol></li><li>第6天，每个女性再次转让到另一个新的小瓶。标签新瓶适当地（以下简称“V3”）。 </li><li>第10天，放弃的 w 1118 </燮&gt;女性。 </li><li>检查V2和V3的后代。女性引入V2（V3）， 例如女性的第一开始产卵V2，后17天之后，第一次检查后，进行13-15天。正是后17天进行第二次检查。这个时间框架构成安全上的时间边界，保证没有第二代相同的小瓶。两次检查防止过度拥挤，同时有利于后代的计数。 <ol><li> 17天，检查v1和确保有红眼睛的后代目前，如果没有标注相应的小瓶。白色眼睛的后代的存在下，表示不是处女雌性而没有后代表明，配合与参考或男性实验并没有出现在初始配合时。 </li><li>第17天，执行第一次检查V2。麻醉后代出现v2中的CO 2和眼睛的颜色和性别对其进行排序。父亲由第一和秒的记录后代号码第二个位图6显示了预期的在每个配合计划的后代的表型。在这个特定的实验14中 ，可以明确地分配唯一的女性的后代，作为参考或实验男性与配，因此只有从女儿的信息可以被用于计算P 2。如果与其他标志物，雄性后代侍可以明确分配，后代数量的男女双方可以用在下游计算。丢弃的后代，但，保持V2第二次检查。 </li><li>第20天，按2.5.2在v2中新涌现出的后代，然后丢弃药瓶。 </li><li>第20天，检查第三版首次出现了后代，并按照步骤2.5.2。 </li><li>第23天，继续在V3中新涌现出的后代2.5.3。 </li></ol></li></ol>注1：由于潜在的P分数膨胀的效果，可能有多次交配（2.2.2和2.2.3），交配可以被限制在一个特定的时间帧。将依赖于频率与基因型的女性和男性使用remating的时间框架。多次交配的概率，特别是减少在夜间11。 注2：不要加，因为这可能会导致潜在的过度生长的酵母成蝇的当数低的问题，是由于活酵母颗粒。 3。数据分析<ol><li>录制的后代计数应该在适当的组织容易视觉检查和高效的分析，用合适的统计软件包（ 如 JMP SAS研究所）或免费的基于Web的工具（ 如 <a href="http://www.vassarstats.net/" target="_blank">http://vassarstats.net/</a> ）。 </li><li>精子的竞争能力。添加v2和v3的罪名。雌蝇引起没有或非常有限的后代（ 如 &lt;10），在手术过程中死亡，只有成功insemina泰德由一个，两个男性被视为无信息的，被排除在任何下游的统计分析（2.5.1和表2）。计算P 2的得分为每个翔实的女性。 </li><li>测试实验的男性相比，P 2值之间是否有统计学上的显着差异。这可以通过使用参数（ 例如 Tukey的HSD）或非参数（ 例如钢Dwass）的测试，这取决于几个因素，包括的P 2值的分布的方差和均值之间的依赖关系的歪斜。通常适用于角度变换参数测试31的使用之前，如P 2的比例。 </li><li>交配率差额（可选）。对于每一个实验的男性类型，计算双重交配的女性和那些只交配与男性（参考男性的罪行检测和实验的男性在d法）efense。使用双尾Fisher精确检验（可在<a href="http://www.langsrud.com/fisher.htm" target="_blank">http://www.langsrud.com/fisher.htm</a> ），确定两种类型的实验男性之间是否有统计学上的显着差异。 </li><li>性别比（可选）。对于每个实验的男性，使用卡方检验，以确定是否偏离预期的1:1的比例从每个女性后代的性别比例。 </li></ol>

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没有利益冲突的声明。

我们已经描述了实验设计，评估基因上不同的差异的相对贡献D.雄性果蝇的后代控制的双交配实验7,8。已经这样做在假设有遗传因素影响精子的竞争能力，已经示出的相似方法虽然适用于防御吸附试验（ 图1）内的罪行检测的上下文中。这种实验的设计可以进行修改，以测试如女性基因型32的影响等方面的侍成功。可以合并的修改的一个例子是直接监测交?...

由于杰夫·帕克指出精子竞争的患病率在昆虫和进化的影响2，一股在果蝇和其他物种的研究，这种现象在许多不同的层面上试图摆脱一些光。感兴趣的领域的一些例子已经及其变化调查9,10自然种群，其遗传结构和潜在的遗传因素的相关性11-14，其作用是在推动两性之间的协同进化15,16。在D中专门的精子贮藏器官的能力有限，一对精囊和的精液插座6,17，雌性果蝇，有助于从不同男性的精子竞争。约1,500精子在交配过程中的女性，但只有约500，可容纳提到的器官18,19。在实验室中，控制双交配实验中涉及的参考男性和一个或多个感兴趣的男性，已被广泛用于评估精子的竞争能力7,8。 精子的竞争能力估计为双交配实验中实验的男性后代总量父系后代的比例， 即从实验和参考男性。精子的竞争能力，由两部分组成，他们每个人在一个单独的试验评估。罪检测，实验的男性的精子，以取代从第一男性的精子的能力， 即参考男性，评估。相反，在防守测定，实验男性精子的能力抵抗位移或减少参考男性的精子受精成功进行了评价。根据法，进攻或防卫的类型，估计精子的竞争能力，通过分数P 1，P 2。 P1和P 2只能取0和1之间的值。中间值通常被解释为间接证据的精子混合，这意味着一个生理情况下涉及直接的精子竞争。同样的理由也可以解释为，极端值强差的精子竞争能力的证据。早期研究表明，在D P 2 果蝇超过0.8，随着时间两者之间的交配延长7。此相同的实验设计已被用于在其他果蝇 ，P 2是常用的统计研究，以评估精子竞争力20。对于大多数种，菌株的P 2值高于0.6 21。然而，不同男性的精子之间的直接竞争无关的其他一些机制可以产生相同的分数（见讨论）。 ð通过使用由所述第一或第二男性与配istinguishing后代能够容易识别的标记。在早期的研究中，男性在亚致死剂量的照射，例如，X射线，使得几乎所有的卵子照射的精子受精失败孵化7。随后，基因突变改变眼部色素沉着或机翼形状是最常用的标记。前者的例子是突变体重 （ 棕色）9，CN（朱砂 ）22 和 w（ 白色 ）23，而突变循环 （ 卷发 ）24对应的第二个不同的表型，这些突变，已被合并在同一个人， 如CN体重 。程度较轻，25位酶和微26,27与已知的遗传模式也被使用。 设计的实验测试的差异这里描述的精子竞争能力如下实质上是克拉克等人 9。来自这些实验结果给出的信息仅差分侍正在审议的实验的男性类型。检测也作出拨备健身14,28和精子的可视化技术24受精后的差异使精子能力的差异被解释为在P 1（或P 2）破门得分的差异。 图1概括了双方的进攻和防御检测的理由。为了说明物流过程中的罪行进行的实验在D果蝇 14将进行详细说明。这种特殊的罪行检测被用于测试可衡量效果的精子特异性基因家族动力蛋白中间链（SDIC）对精子的竞争能力。所有成员s的这个基因家族位于X染色体上的串联。淘汰赛男性产生通过删除国投集群。由于删除的板块还包括必要的的基因短翼（SW），研究的目的是评估的相关性， 国投 ， 国投-SW缺失的男性携带被救出由转基因副本SW（标志为P {SW} 2号染色体上，还进行了一个小型记者白基因）。眼睛颜色被用来作为一个可见的标记亲子鉴定。所有的苍蝇在白色突变的背景，除了那些从应变俄勒冈-R，它被用来作为参考男性。

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Amber latex tubing</td>
			<td>VWR</td>
			<td>62996-473</td>
			<td>1/4 inch ID, 1/6 inch Wall</td>
		 </tr>
		 <tr>
			<td>1 ml graduated XL filter tips</td>
			<td>USA Scientific</td>
			<td>1126-7810</td>
			<td>Any 1 ml pipette tip can be used but semi-transparent and long tips are better as a fly receiver. If filter tips are used, poke out and discard the filter using a needle after the tip end is trimmed</td>
		 </tr>
		 <tr>
			<td>Parafilm</td>
			<td>Sigma</td>
			<td>P-7793</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mesh Fabric</td>
			<td></td>
			<td></td>
			<td>Thin and smooth</td>
		 </tr>
		 <tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>S6D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CO2 Tank</td>
			<td>Airgas</td>
			<td>CD-50</td>
			<td>Use FlyNap (Carolina Biological Supply Company) as an alternative if CO2 is not available</td>
		 </tr>
		 <tr>
			<td>FlyStuff Foot Valve, complete system</td>
			<td>Genesee Scientific</td>
			<td>59-121C</td>
			<td>Not necessary if FlyNap is used</td>
		 </tr>
		 <tr>
			<td>Plastic vials</td>
			<td>Genesee Scientific</td>
			<td>32-109</td>
			<td>Come with carboard trays that can be reused for holding vials</td>
		 </tr>
		 <tr>
			<td>Cotton balls</td>
			<td>Fisher Scientific</td>
			<td>AS-212</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Active dry yeast</td>
			<td>Red Star</td>
			<td></td>
			<td>Found in general grocery store</td>
		 </tr>
		 <tr>
			<td>Sharpie markers</td>
			<td></td>
			<td></td>
			<td>Different colors may be used for marking different genotypes</td>
		 </tr>
		</tbody>
 </table>

assessing differences in sperm competitive ability in drosophila

卵子受精的同种雄性之间的竞争性选择的机制之一， 即选择上运行的数量最大化，成功交配事件，而不是最大化的生存和活力1。精子竞争代表相同的女2，他们的精子是在时间和空间上的巧合交配后，雄性之间的竞争。这一现象已在多种植物和动物物种的3。例如，野生捕捞D.果蝇女性通常含有精子2-3名男性4。精子被储存在专门机关与有限的存储容量，这可能会导致直接的竞争，从不同男性的精子2,5。 权益（实验的男性类型）不同的男性的精子竞争能力的比较，通过控制双交配实验技术已执行在实验室6,7日ts。简单地说，一个单一的女性露出两个不同的男性连续的，一个实验的男性和一个交叉配合参考男性。相同的交配计划是随后使用其他实验的男性类型，从而促进他们的精子的竞争能力，通过一个共同的参考间接比较。父亲的实验和参考男性的个人的分数是确定使用的标记，它允许一个估计精子的竞争能力，用简单的数学表达式7,8。此外，精子的竞争能力，可以估算在两种不同的情况，根据实验的男性是否是第二或第一的队友（进攻和防守法，分别）9，被认为是反映不同的能力属性。 在这里，我们描述了一种方法，有助于询问不同的遗传因素的作用公认的基础吨他现象D.精子的竞争能力果蝇 。

表2总结了两个罪行实验（化验1和2）的一些主要特征，其中D。 （I和II型），分别的功能国投集群与无果蝇实验的男性相比，14。考虑后遇到一些重复的不同发病率58-83％的女性被认为是信息，因此他们的后代可以用于计算P 2侍计数。非参数检验的结果是一致的，男性精子的竞争能力，而不的国投集群（II型）相比，男性与完整的集群（I型<su...

Watch this Scientific Journal Video about Assessing Differences in Sperm Competitive Ability in Drosophila at JoVE.com

Assessing Differences in Sperm Competitive Ability in Drosophila

差分精子之间的竞争能力<em&gt;果蝇</em&gt;男性不同的基因型，可以通过双交配实验确定。这些实验中的每一个都涉及到一个感兴趣的男性和一个参​​考男性。后代中易于识别的标记允许每个男性父亲的个人分数的推论。

评估精子竞争能力的差异<em&gt;果蝇</em

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the ...

assessing-differences-in-sperm-competitive-ability-in-drosophila

Research

JoVE Journal

Biology

Assessing Differences in Sperm Competitive Ability in Drosophila

14.5K 次观看.  University of California, Irvine. 差分精子之间的竞争能力果蝇男性不同的基因型，可以通过双交配实验确定。这些实验中的每一个都涉及到一个感兴趣的男性和一个参​​考男性。后代中易于识别的标记允许每个男性父亲的个人分数的推论。

视频: Assessing Differences in Sperm Competitive Ability in Drosophila

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

荧光原位杂交（FISH）的议定书“在人类精子

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

科研

生物学

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

技术成像钙 2 +信号在人类精子

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

分离和<em在体外激活线虫精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

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  ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

可视化和跟踪活果蝇黑色素细胞卵室的内源性 mRNA

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

中通量筛选法评估对精子中 ca2 +信号和反应的影响

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to recognize a wide range of particles and destroy microbial pathogens. As organisms age, this process begins to decline, yet little is known about the underlying mechanisms or the genetic basis of immunosenescence. Here, an injection based in vivo phagocytosis assay is used to assess age related changes in different aspects of phagocytosis, such as binding, engulfment, and degradation of internalized particles, by quantifying phagocytic events in hemocytes in adult Drosophila. Drosophila melanogaster has become an ideal model to investigate age related changes in innate immune function for many reasons. For one, many genetic components and functions of the innate immune response, including phagocytosis, are evolutionarily conserved between Drosophila and mammals. Because of that, results obtained from using this protocol are likely to be widely relevant to understanding the age related changes in immune function in a variety of organisms. Additionally, we note that this method provides quantitative estimates of hemocyte phagocytic ability, which could be useful for a variety of research topics, and need not be limited to studies of aging.

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

This protocol describes an in vivo assay of phagocytosis used to assess and quantify the ability of young and aged Drosophila melanogaster hemocytes to phagocytose bacteria.

使用 Vivo 法戈西西亚赛评估成人嗜血杆菌黑色粒细胞的年龄特异性噬细胞能力

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

解剖、固定和可视化 果蝇 蛹

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...