The authors thank David Larson for developing the original protocol for imaging the live Drosophila pupal eye. We thank our colleagues Richard Carthew, Shoichiro Tsukita and Matthew Freeman, who developed the transgenic flies that we combined to generate our live-imaging fly lines. Brittany Baldwin-Hunter gave helpful comments on the manuscript. This work was supported by start-up funds awarded to Ruth Johnson by Wesleyan University.

图3示出的实验程序的概要。 1.组织准备<ol><li>要确定目标基因的表达修饰的后果，双份以下的交叉，在25℃保持C：UAS-转基因 （男性）X GMR-GAL4; UAS-α-猫：GFP，UBI-DE-加元：GFP; + / SM5-TM6b（处女女）注：要获得控制组织交叉UAS-lacZ的男性为GMR-GAL4; UAS-α-猫：GFP，UBI-DE-加元：女性GFP。 β半乳糖苷酶产生在视网膜中的细胞行为没有缺陷。 </li><li>为了提高转基因的RNAi的疗效，跨UAS-RNA干扰雄性GMR-GAL4，UAS-DCR-2; UAS-α-猫：GFP，UBI-DE-加元：GFP; + / SM5-TM6b处女的女性。 注：在细胞行为表达异位DCR-2已经观察到偶尔的缺陷在视网膜（数据未示出）。警惕，建议如果使用这种方法。 </li><li> S选白色预蛹适当的基因型和地点1.5 ml离心管。如果蛹表达DCR-2，选择男或女蛹：UAS-DCR-2，X染色体上的插入，表达是男性更强。蛹应在0小时的APF蛹壳的色素前绝育手术 - 男性性腺如在蛹（大约三分之一从后）的侧面大半透明地球仪可见。 </li><li>孵育含蛹，在一个干净的塑料提示框，在25℃的离心管。为了防止蛹的地方干燥10 平方厘米片组织，浸泡在蒸馏水中，将尖端盒。 注意：如果湿度在尖部 - 框变得太高蛹壳可能会变得潮湿和难以在后续步骤中进行解剖。 </li><li>孵育适当的时间。为了捕捉构图眼有关的细胞行为，准备蛹成像约17小时APF，20小时APF或24小时APF.查看时，特定的细胞行为是最好的观察作进一步的讨论代表性的成果。 </li><li>将一块新鲜的双面胶带上一个黑色的SYLGARD解剖盘。定位蛹，背侧，与坚持双面胶带（ 图1A）蛹的头。尝试不坚持蛹的胸部和腹部地区的双面胶带。用钳子小心地提起并取出鳃盖，露出蛹头。沿蛹壳的侧撕到区域露出围绕一只眼睛。 </li><li>轻轻地取下胶带蛹。如果蛹仍粘附的，施加一个小体积的蒸馏水，以结蛹壳和带之间，以削弱的粘合。 </li></ol> 2.安装<ol><li>构造从吸墨纸小15毫米2帧和冲头在middlethat一个孔为5.5毫米的直径（ 图1B）。浸泡在蒸馏水中并将其放置在中心的显微镜载玻片。使用30毫升注射器，挤压出凡士林的均匀环以围绕吸墨纸帧。确保凡士林环稍高于蛹的宽度和该环的直径略微大于盖玻片的宽度小于高。 </li><li>添加一个小珠凡士林直接到幻灯片，在冲压成的吸水纸（ 图1C）.Carefully安排蛹，在其一侧洞口，由凡士林珠（ 图1D）的支持。 </li><li>放置盖玻片上凡士林环的顶部，使得其接触上述眼球神经上皮（ 图1D）的上皮。轻轻压缩编制以密封的凡士林盖玻片并生成蛹眼睛区域和盖玻片之间的小平面的接触表面。 </li></ol> 3.荧光成像<ol><li>图像使用蛹准备荧光显微镜。每7分钟捕获通过眼睛神经上皮的心尖域，在粘着路口的区域连续切片。 注：a）适当的串行部分通常是4-5元的z栈包括0.2微米的Z-步骤。 b）在上皮细胞的拓扑结构，包括轻度颠簸和不规则的褶皱，可能使其具有挑战性的收购聚焦图像组织大型的区域。而成像，人们可以发现感兴趣的区域的一部分，以在聚焦，和相邻区域是失焦。包括蒸馏水蛹眼睛和盖玻片之间的小液滴可以消除一些急性组织褶皱而不是蛹眼形态的早期阶段的轻度不均匀拓扑特征。在这些情况下，过采样的组织通过扩展Z堆叠直到在焦切片获取整个字段。三）捕获连续切片，每7分钟应使活成像为3至4个小时没有显著热镀ction在GFP荧光可能危害图像质量的强度。较短的时间间隔可以降低成像的总时间。 </li><li>继续成像为3至4个小时。不要使用自动对焦和时间的函数，是许多荧光显微镜，因为蛹增长和血淋巴抽移动视网膜的位置警惕再聚焦，需要每14-21分钟。注：该成像超过4时间可能会减缓或停滞眼睛的形态。 </li><li>使用适当的反卷积软件，以减少背景和增强连续切片图像的对比度。对于每一个Z堆栈文件，请在LAS AF软件以下（见表材料）：导航到工具面板中，选择3D反卷积，然后单击应用。 </li><li>生成每个卷积堆栈文件的最大投影（MP）影像：导航到工具面板中，选择3D投影，然后单击应用。 注：最大投影的算法可能无法生成一个统一的LY聚焦图像用于组织已经被过取样。一个技术磨砺出焦地区在步骤5.2概述。 </li></ol> 4.成像后蛹救援和表型验证<ol><li>为了解决文物是否引进或成像过程中损害了蛹，小心地取出从成像钻机蛹：使用镊子取出盖玻片和蛹转移到食物的干净的小瓶。储存在室温或25℃，直到成年果蝇出现。 </li><li>仔细检查成人眼睛的表型，并比较成蝇新兴从原始蝇交叉的眼睛。 </li></ol> 5.图像处理<ol><li>自动图像对齐方式： 注：本次现场果蝇组织将转向和整个成像过程成长。因此，图像的中心聚集在连续的时间点可能不对应于同一点，在果蝇的组织。在另外的整个视网膜盘片旋转21〜23小时之间APF约30°。为了突出单个细胞的行为和减少因有机体的生长杂念，调整每个MP image.While这可以手动执行，这里使用的图像编辑软件（ 见表材料 ）有内置的算法，可以加快这一进程。 <ol><li>从主菜单，打开脚本，选择将文件加载到堆栈的文件选项卡。 </li><li>导入MP图像文件转换加载图层面板中，选择确定。确保尝试自动对齐源图像选项没有被选中。 注意：如果选择此选项，该软件可使用比对算法的扭曲的图像数据。 </li><li>一旦所有的MP文件已加载到图层面板中，确保所有的图像都按时间顺序，例如，最早的时间点是在层堆栈的顶部。如果层不按正确的顺序，拖放他们，直到他们被命令正确。 </li><li>从主菜单中的编辑选项卡中选择自动对齐图层。选择重新定位，然后单击确定。 </li><li>如果自动对齐算法不能帧定位到相关的焦点，让使用移动工具小幅调整。 </li></ol></li><li>复合锐化： 注：离焦的初始的MP图像的区域可以通过“裁剪”在LAS AF软件的相应解卷积堆栈进行锐化。裁剪一叠文件允许一个手动限制经受最大投影算法的z堆栈的间隔。 <ol><li>找到在工具面板中的裁剪功能（进程选项卡下）。手动限制的最初和最后的片，使得它们仅跨越最初失焦区域内的粘合带。单击应用生成针对该区域优化的新的堆栈文件。 </li><li>生成一个新的投影最大的本地优化堆栈和出口为TIFF文件。 </li><li>在日Ë图像编辑软件（见表材料），开放式脚本（位于主菜单中的文件选项卡），然后选择将文件加载到堆栈。 </li><li>导入特定时间点进入加载图层面板中的初始MP图像文件，并在本地优化的MP文件，并选择确定。确保尝试自动对齐源图像选项没有被选中。 </li><li>选择这两个图层的图层窗格，然后选择自动混合图层（位于主菜单中的编辑选项卡）来自动屏蔽两个图像的适当区域，产生一个统一的，集中的视网膜领域。保存此合成图像为TIFF文件。 </li><li>重复步骤5.2.1到5.2.5的所有不理想的MP图像。 </li></ol></li><li>进一步的调整：旋转，裁剪和调整图层电影的水平： <ol><li>与所有层的选择，使用变换工具（编辑&gt;自由变换），旋转图像，使视网膜的背腹轴一致到电影帧的y轴。 </li><li>如果需要的话，可使用裁剪工具，以减少视场成所需的尺寸和形状。 </li><li>使用电平调整（的各个帧），以帮助提高细胞膜和细胞体之间的对比。 </li><li>为文体的目的，并强调特定的细胞的行为，引入假色突出显示的每个帧中的兴趣点。 </li></ol></li><li>动画：转换图像成电影： <ol><li>从主菜单中的Windows选项卡，打开动画面板。选择做自层帧从位于动画面板的右上角的菜单。 </li><li>注意，这些层被添加到动画窗格按顺序从堆栈的底部开始。为了扭转帧的顺序，首先选择所有的帧，然后从动画面板菜单中选择逆向框架。 </li><li>使用帧拇指下方的下拉菜单中选择用于每个帧一帧的延迟时间钉子。 注：帧延迟为电影1至4本文介绍的是0.8秒。 </li><li>在主菜单中，打开导出的文件选项卡，然后选择渲染视​​频。选择QuickTime导出下文件选项，然后选择所需的视频格式。在渲染选项设置帧速率自定义，进入15帧速率，并单击渲染。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

野生型的发展（上图）的描述形成的基础，在RNAi的图案事件或过表达基因型比较。精确确定哪些细胞行为是由感兴趣的蛋白质调节的时活组织发育的比较是非常宝贵的。此外，并没有在这里描述的，活细胞成像使得人们能够使所关注的基因的作用的定性和/或定量描述的事件模式的眼睛。经30-32小时APF最色素细胞已经获得稳定的位置和凋亡已经从视网膜现场修剪多余的细胞。在此之后，仅微妙的变...

化合物果蝇眼睛的特征在于它的〜750小眼由一个蜂窝状晶格附件色素细胞4,5的隔开的定型装置。局部细胞运动，细胞生长，改变细胞形状和细胞凋亡：这些色素细胞是由事件的协调组合图案化。这种上皮现场可视化允许一个研究在生理学相关和泰然自若的三维环境支撑这些事件的分子机制。 相较于以前的协议6,7，这里概述的技术结合用于稳定外部组织的运动，不能从成像过程耦合的有效方法。这种方法提高了在显影果蝇蛹眼上皮细胞的行为的研究- ，生长，旋转的组织，并移过成像的过程。此外，运动稳定技术去这里划线将是在无关的运动发生其他上下文研究细胞是有用的。 可视化细胞界限在果蝇视网膜领域，产生了转基因飞线表达UBI-DE-钙粘蛋白：GFP和UAS-α-catenin的：GFP在眼部专用的驱动程序GMR-GAL4 1-3的控制权。使用两个GFP标记膜标记允许小区边界的在较低强度的光的可视化。本上重复暴露于高能量波长的光，使增加的帧速率和电影的持续时间最小化组织损伤和光漂白。为了提高转基因的RNAi，UAS-DCR-2也被纳入到第二飞线8疗效。 在以前协议的第三次更新，这是很容易组装在大多数实验室一个简单的成像钻机描述。该装置避免了要求有一个专门的成像řIG一所大学的“加工车间”或类似的服务产生。该成像钻机类似于用于其它图像蛹组织9,10。 这里介绍的是可以用来直接评估，经过17至42小时puparium形成（小时APF）有助于眼睛的图案来自〜的形态发生事件的简单的活细胞成像协议。具体地，该协议使得人们能够确定蛹发育过程中修饰基因表达的后果。

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			<td height="21" style="height:21px;width:263px;">Name of Material/ Equipment</td>
			<td style="width:124px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">GMR-GAL4; UAS-a-cat:GFP, ubi-DE-Cadherin:GFP ; + / SM5-TM6b</td>
			<td>Available on request from authors</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">UAS-dcr-2; UAS-a-cat:GFP, ubi-DE-Cadherin:GFP; + / SM5-TM6b&#160;</td>
			<td>Available on request from authors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Scotch double stick tape</td>
			<td style="width:124px;">3M</td>
			<td style="width:119px;">665</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Black Sylgard dissection dish</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>Fill glass petri dish to rim with Sylgard (colored black with finely-ground charcoal powder) or Sylgard 170; leave at room temperature for 24-48 h to polymerize</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Sylgard</td>
			<td style="width:124px;">Dow Corning</td>
			<td style="width:119px;">SYLG184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Sylgard (Black)</td>
			<td style="width:124px;">Dow Corning</td>
			<td style="width:119px;">SYLG170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Glass petri dish</td>
			<td style="width:124px;">Corning</td>
			<td style="width:119px;">7220-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">25 x 75 x 1 mm glass microscope slide</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">12-550-413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">22 x 40 mm glass coverslip</td>
			<td style="width:124px;">VWR</td>
			<td>48393-172</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Forceps&#160;</td>
			<td style="width:124px;">Fine Science Tools</td>
			<td style="width:119px;">91150-20</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Whatman 3mm Chromatography Paper</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">05-713-336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Vaseline</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">19-086-291</td>
			<td>Or purchase at local pharmacy.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">30ml syringe</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">S7510-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Adobe Photoshop CS5</td>
			<td style="width:124px;">Adobe</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Leica TCS SP5 DM microscope</td>
			<td style="width:124px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">LAS AF Version 2.6.0.7266 microscope software</td>
			<td style="width:124px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

live-imaging of the drosophila pupal eye

果蝇蛹发展的内在过程可以转移和扭曲眼球上皮细胞的方式，使各个细胞的行为很难在活细胞成像跟踪。这些方法包括：视网膜旋转，细胞生长和有机体的运动。此外，在上皮的拓扑，包括细微凸块和凹凸褶皱经常引入作为蛹被用于成像准备，使其具有挑战性获得在单个焦平面在焦像以上几个小眼。这里的工作流程概述了补救这些问题，让细胞过程中果蝇蛹眼睛的发育很容易分析。适当上演蛹被布置在可容易地装配在大多数实验室成像钻机泛素- DE-钙粘蛋白：GFP和GMR-GAL4驱动的UAS-α连环蛋白：GFP被用于可视化小区边界在眼上皮1 -3。反褶积之后是施加到在多个焦平面捕获荧光图像，产生用于每个时间点的最大的投影图像，并使用图像编辑软件增强。比对算法用于快速稳定多余的动作，使得单个细胞的行为更容易追踪。

实时成像蛹眼睛是一个成功的策略，以观察细胞的行为，有助于神经上皮的图案。特定蛋白质的作用，可以通过表达眼睛的发育过程中修改的蛋白质水平的转基因可以容易地评估。要做到这一点的GMR-GAL4用于驱动所述形态发生沟后面的转基因表达。这提供了不扰动早期事件，在头两个幼虫龄建立眼场的优点。此外，许多的RNAi的转基因需要显著的时间来有效地降低蛋白质的水平（数据未示出?...

Watch this Scientific Journal Video about Live-imaging of the Drosophila Pupal Eye at JoVE.com

Live-imaging of the Drosophila Pupal Eye

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

实时成像<em&gt;果蝇</em&gt;蛹眼

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

Research

JoVE Journal

Developmental Biology

Live-imaging of the Drosophila Pupal Eye

9.3K 次观看.  Wesleyan University. This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

视频: Live-imaging of the Drosophila Pupal Eye

Long-term Live Imaging of Drosophila Eye Disc

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

Long-term Live Imaging of Drosophila Eye Disc

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

长期的实时成像<em&gt;果蝇</em&gt;眼睛光盘

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

科研

发育生物学

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells named wing imaginal discs formed during embryonic development. In the larval stages the imaginal discs grow, increasing its number of cells and forming monolayered epithelial&#160;structures. Inside the pupal case, the imaginal discs bud out and fold into bilayers along a line that becomes the future margin of the wing. During this process, the longitudinal primodia veins originate vein cells on the prospective dorsal and ventral surfaces of the wing. During the pupal stage the stripes of vein cells of each surface communicate in order to generate tight tubes; at the same time, the cross-veins begin their formation.
With the help of appropriate molecular markers, it is possible to identify the major elements composing the wing during its development. For this reason, the ability to accurately detect transcripts or proteins in this structure is critical for studying their abundance and localization related to the development process of the wing.
The procedure described here focuses on manipulating pupal wings, providing detailed instructions on how to dissect the wing during the pupal stage. The dissection of pupal tissue is more difficult to perform than their counterparts in third instar larvae. This is why this approach was developed, to obtain rapid and efficient high quality samples. Details of how to immunostain and mount these wing samples, to allow the visualization of proteins or cell components, are provided in the protocol. With little expertise it is possible to collect 8-10 high quality pupal wings in a short amount of time.

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

The ability to accurately detect transcripts or proteins in Drosophila tissues is critical for studying their abundance and localization related to the development process. Here is the description of a straightforward procedure to dissect pupal wings. These wings can be used as samples in several techniques (immunohistochemistry, PCR assay, etc.).

一种快速高效的解剖蛹翅的方法, 适用于 Immunodetections 或 PCR 检测

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

果蝇蛹卵巢的解剖与染色

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

嗜酸性粒植物视网膜免疫组织化学、西方分析和 Rna 分离的解剖

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

果蝇胚胎肌肉收缩的实时成像和分析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

解冻、培养和冷冻保存的嗜酸性粒细胞系

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

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

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

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

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

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

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

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

用于活、完整组织中细胞分部分析的果蝇拉瓦尔和普帕尔睾试验的制备

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...