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

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

1. 解剖前一天的准备
<ol><li>电解磨钨针24 ，使得到的直径约为0.03毫米。将供电电压调节至约14 V，并使用3.3 M NaOH。锐化时间不应超过 1 或 2 分钟。 注意：NaOH具有高度腐蚀性，与皮肤接触会导致灼伤。处理时戴上手套和护目镜，并在通风橱内工作。 注意：使用后，将NaOH储存在聚丙烯猎鹰管中。</li>
	<li>制作准备好的成像介质。在15 mL锥形管中，将4.25 mL施耐德培养基与750μL?...

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

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

<table>
	<tbody>
		<tr>
			<td>Alfa Aesar&#160;Tungsten wire</td>
			<td>Fisher Scientific</td>
			<td>AA10408G6</td>
			<td>0.25mm (0.01 in.) dia., 99.95% (metals basis)</td>
		</tr>
		<tr>
			<td>D. melanogaster: His2Av::mRFP1</td>
			<td>Bloomington Drosophila Stock Center (BDSC)</td>
			<td>FBtp0056035</td>
			<td>Schuh, Lehner, &#38; Heidmann, Current Biology, 2007</td>
		</tr>
		<tr>
			<td>D. melanogaster: nos-lifeact::tdtomato</td>
			<td>Gift from Ruth Lehmann Lab</td>
			<td></td>
			<td>Lin, Luo, &#38; Lehmann, Nature Communications, 2020: nos5&#39;- Lifeact-tdtomato p2a tdkatushka2 Caax nos3&#39;</td>
		</tr>
		<tr>
			<td>D. melanogaster: P-Dsix4-eGFP::Moesin</td>
			<td></td>
			<td>FBtp0083398</td>
			<td>Sano et al., PLoS One, 2012</td>
		</tr>
		<tr>
			<td>Diamond-tipped knife</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch</td>
			<td>665</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GIBCO</td>
			<td>10082</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging dish</td>
			<td>MatTek</td>
			<td>P35GC-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>MetaMorph Microscopy Automation and Image Analysis Software v7.8.4.0</td>
		</tr>
		<tr>
			<td>Insulin, bovine</td>
			<td>Sigma</td>
			<td>l0516</td>
			<td>Store aliquots at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Fisher Scientific</td>
			<td>08-955</td>
			<td></td>
		</tr>
		<tr>
			<td>Nytex basket</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Ringer&#39;s solution</td>
			<td></td>
			<td></td>
			<td>2 mM MgCl2, 2 mM CaCl2, 130 mM NaCl, 5mM KCl, 36 mM Sucrose, 5mM Hepe&#8217;s Buffer; adjusted with NaOH until pH of 7.3 is achieved</td>
		</tr>
		<tr>
			<td>Schneider&#39;s Insect Media</td>
			<td>GIBCO</td>
			<td>21720-024</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and live-imaging of the late embryonic drosophila gonad

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

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

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

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

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

晚期胚胎 果蝇 性腺的解剖和活体成像

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

Research

JoVE Journal

Developmental Biology

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

大规模的斑马鱼胚胎的心脏解剖的转录分析

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

科研

发育生物学

Live-imaging of the Drosophila Pupal Eye

Live-imaging of the Drosophila Pupal Eye

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 ...

Dissection and Culture of Mouse Embryonic Kidney

小鼠胚胎肾的解剖与培养

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Long-term Live Imaging of Drosophila Eye Disc

Long-term Live Imaging of Drosophila Eye Disc

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 ...

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

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

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 Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Dissection and Staining of Drosophila Pupal Ovaries

Dissection and Staining of Drosophila 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

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

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

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

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 ...

晚期胚胎 果蝇 性腺的解剖和活体成像

晚期胚胎果蝇性腺的解剖和活体成像