We appreciate and would like to thank the Howard Hughes Medical Institute for the HHMI Professor award to U.B. which made this project possible. We thank the college at the University of California, Los Angeles for providing facilities and teaching infrastructure support for this work. The work was also supported with funding from Midwestern University and a generous donation from the Charity Fidelity Gift Fund. We thank John VandenBrooks for comments on the manuscript and Krista Pearman for her technical assistance.

这个过程是2天的过程。 第1天（2小时+解剖时） 1.蛹眼睛光盘解剖<ol><li>选择一个蛹解剖。 注：蛹被解剖的年龄将通过实验需要来确定。然而，如果检查细胞形态，这是经常做在puparium形成（APF）后42小时，在25℃，这是在视频中所示的蛹的年龄。收集白色蛹（视为0小时，APF）与润湿画笔并安排它们按时间顺序（基于收集时间）在保持于25℃的新飞食品小瓶中。解剖老龄蛹在此相同的时间顺序，以保持蛹尽可能接近之间的时间差异。 </li><li>转移蛹成一滴磷酸盐缓冲盐水（PBS，pH 7.4中，见材料配方）的聚硅氧烷解剖板。 </li><li>握住蛹壳钳和皮尔斯的顶部，并打开升蛹壳有一对锋利的钳的奥尔部分。从在新成立的孔的情况下取出蛹。 </li><li>做一个对角用剪刀剪，中胸部，在蛹。除去碎屑或蛹头转移到PBS的新下降。清洁用吸管鼓风机管（见材料和图1说明），并根据需要增加额外的PBS。一定要避免脱水。 </li><li>牢固地夹住蛹头角质层有一对镊子的顶端。使用吹风管的前端，轻轻推蛹壳挤出的脑和其他组织，包括眼的光盘，从蛹壳中。一定要避免角质层内抓住任何组织。 </li><li>使用吹风管充满PBS，轻轻吹脂肪人体组织和​​其他碎屑头的情况下清楚地分开，并确定了大脑和眼睛的光盘。 注：保持附着在蛹头壳体的大脑将允许研究者使用他广告的情况下，作为所述组织的未来操纵一个“把手”，而不会损坏眼睛光盘或脑。然而，这是常见的撞出从头部壳体的脑和眼睛的光盘，这是不利于整体过程和结果。往往需要吹和推成功地从内蛹头壳体和使它们清楚地从周围组织中分离提取出的眼盘和大脑的多次尝试。清洁器和多个分离的人能得到从周围组织中的眼睛的光盘将有助于防止外部组织从固定到眼睛的光盘。 </li><li>下面的夹层，立即将组织用0.5ml的修复溶液（见材料）转移到3以及玻璃皿置于冰上。浮在组织几分钟（通常花费解剖下一组眼盘的时间）的修复溶液的表面上。 注 ：如果这不是第一眼盘在这一轮的清扫，completel被解剖Ÿ淹没以前的眼睛光盘进入修复解决方案。如果执行特殊染色程序（ 例如 ，活性氧，溶酶体等），这必须被固定之前完成。为了做到这一点，请将眼球解剖盘在冰冷的PBS之前，用适当的染色方案继续进行。 </li><li>以下夹层的所有眼光盘和浸没被解剖，将3以及玻璃盘到一个摇杆和岩石在室温下搅拌30分钟。 </li></ol> 2.免疫染色<ol><li>小心地取出修复溶液并加入0.5毫升的PBS与0.3％的Triton X-100（0.3％的PBT，见材料）和洗组织上的摇杆10分钟。总共4次洗涤（每次10分钟）重复此步骤3次以上。 注意：小心除去从孔中的溶液，可在解剖显微镜，以防止组织损伤或损失下进行。该溶液可以小心地用移液管吸出;我们更细tippeD，一次性移液管的已夷为平地，以减少组织吸的几率开幕。 </li><li>在第4次洗涤结束后，取出清洗和添加0.5毫升块溶液（见材料）到井和温育30分钟。 注：此时间段可以根据自己的需要进行扩展。 </li><li>在这30分钟块结束时，移液管加入10μl初级抗体溶液（稀释在方框溶液）放入井在微孔板的合适数量。 注：微孔板的一个井将容纳约4对附着在蛹壳眼睛光盘或8路隔离对眼睛光盘附带的大脑。在此协议为代表的数据所使用的一抗是抗 - 磷酸（1：500稀释）和抗剪切（1：200稀释）。 </li><li>转移的解剖组织成微孔板孔中填充了一抗溶液从3以及玻璃皿中。 </li><li>地方在润湿的纸巾的微孔板（防止脱水）在空吸管尖盒（或其他类似的容器），并储存在4℃下过夜。 </li></ol>第2天（4小时+的安装时间） <ol start="6"><li>传送从微孔板组织到一个新的3孔玻璃培养皿用0.3％的PBT（≈0.5毫升）洗所述组织上的摇杆10分钟。重复此步骤3次以上，总共4次洗涤（每次10分钟） </li><li>除去最后的洗涤和加入0.5毫升座溶液到孔中。阻断30分钟。 注：此时间段可以根据自己的需要进行扩展。 </li><li>从光盖或卸下铝块解决方案，并加入0.5毫升二抗溶液，以井（从适合你的一抗和荧光波长，在稀释液座荧光标记的抗体制成）和保护3以及玻璃盘贴膜孵育至少2小时，在室温温度。 注意：所有的后续步骤应同样不受光线照射。该温育可以扩展到过夜，在4℃下，以类似于第一抗体温育一个设置，但它也可以在覆盖着实验室膜，或​​者，如果抗体保护期望微孔板3以及玻璃盘进行。 </li><li>在抗体温育结束时，除去二抗溶液，并添加0.5毫升0.3％的PBT的孔，并且洗涤在摇臂10分钟。重复，共4次洗涤该步骤（每次10分钟）。 注：如果DAPI染色是期望的，在开始第一次洗涤之前，加入0.5微升的DAPI原液（见材料）与500微升的PBT，将用于对所述第一洗涤液（1：1000稀释）。 </li></ol> 3.安装<ol><li>在最后一次洗涤结束时，安装在滑动用70％的甘油或PBS触角/眼光盘作为介质以除去BR泉和在幻灯片上的一侧上的池其它不想要的组织。 </li><li>小心使用镊子之间的静水压力移动所述组织，或非常轻轻地用钳子前端，并在朝向滑动件的中心柱排队眼光盘。 </li><li>来自各地的组织去除所有多余的甘油或PBS（用实验室的毛细作用擦拭或镊子工作这一点）。 </li><li>放置安装介质的少量（≈10微升）沿着眼睛光盘柱的一侧和放置盖玻片上的组织，从而被散布在安装介质到眼睛上的光盘。 </li><li>让滑坐为1至2分钟，以允许组织/安装介质散开完全。 </li><li>来自各地的盖玻片的边缘擦拭任何额外的安装介质的切断，并用透明指甲油密封。 </li><li>捕捉利用共聚焦显微镜的眼睛光盘荧光图像。 </li></ol>

<ol>
	<li>Ready, D. F., Hanson, T. E., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Drosophila+retina,+a+neurocrystalline+lattice.">Development of the Drosophila retina, a neurocrystalline lattice.</a> Dev Biol. 53 (2), 217-240 (1976).</li><li>Wolff, T., Ready, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beginning+of+pattern+formation+in+the+Drosophila+compound+eye:+the+morphogenetic+furrow+and+the+second+mitotic+wave.">The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave.</a> Development. 113 (3), 841-850 (1991).</li><li>Carthew, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+in+the+Drosophila+eye.">Pattern formation in the Drosophila eye.</a> Curr Opin Genet Dev. 17 (4), 309-313 (2007).</li><li>Bao, S., Cagan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+adhesion+mediated+by+Hibris+and+Roughest+regulates+morphogenesis+and+patterning+in+the+Drosophila+eye.">Preferential adhesion mediated by Hibris and Roughest regulates morphogenesis and patterning in the Drosophila eye.</a> Dev cell. 8 (6), 925-935 (2005).</li><li>Grzeschik, N. A., Knust, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IrreC/rst-mediated+cell+sorting+during+Drosophila+pupal+eye+development+depends+on+proper+localisation+of+DE-cadherin.">IrreC/rst-mediated cell sorting during Drosophila pupal eye development depends on proper localisation of DE-cadherin.</a> Development. 132 (9), 2035-2045 (2005).</li><li>Sprecher, S. G., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switch+of+rhodopsin+expression+in+terminally+differentiated+Drosophila+sensory+neurons.">Switch of rhodopsin expression in terminally differentiated Drosophila sensory neurons.</a> Nature. 454 (7203), 533-537 (2008).</li><li>Zuker, C. S., Cowman, A. F., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+structure+of+a+rhodopsin+gene+from+D.+melanogaster.">Isolation and structure of a rhodopsin gene from D. melanogaster.</a> Cell. 40 (4), 851-858 (1985).</li><li>Voas, M. G., Rebay, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+integration+during+development:+insights+from+the+Drosophila+eye.">Signal integration during development: insights from the Drosophila eye.</a> Dev Dyn. 229 (1), 162-175 (2004).</li><li>Hsiao, H. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+and+immunohistochemistry+of+larval,+pupal+and+adult+Drosophila+retinas.">Dissection and immunohistochemistry of larval, pupal and adult Drosophila retinas.</a> J Vis Exp. , 4347 (2012).</li><li>Call, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomewide+clonal+analysis+of+lethal+mutations+in+the+Drosophila+melanogaster+eye:+comparison+of+the+X+chromosome+and+autosomes.">Genomewide clonal analysis of lethal mutations in the Drosophila melanogaster eye: comparison of the X chromosome and autosomes.</a> Genetics. 177 (2), 689-697 (2007).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery-based+science+education:+functional+genomic+dissection+in+Drosophila+by+undergraduate+researchers.">Discovery-based science education: functional genomic dissection in Drosophila by undergraduate researchers.</a> PLoS Biol. 3 (2), 59 (2005).</li><li>Wolff, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+techniques+for+pupal+and+larval+Drosophila+eyes.">Dissection techniques for pupal and larval Drosophila eyes.</a> CSH Protoc. 2007, (2007).</li><li>Williamson, W. R., Hiesinger, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+developing+and+adult+Drosophila+brains+and+retinae+for+live+imaging.">Preparation of developing and adult Drosophila brains and retinae for live imaging.</a> J Vis Exp. 37, 1936 (2010).</li><li>Xu, T., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues.">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117 (4), 1223-1237 (1993).</li><li>Duffy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAL4+system+in+Drosophila:+a+fly+geneticist's+Swiss+army+knife.">GAL4 system in Drosophila: a fly geneticist's Swiss army knife.</a> Genesis. 34 (1-2), 1-15 (2002).</li></ol>

The authors have nothing to disclose.

While it appears that the process is simple and easy to perform, in reality, this technique requires a great deal of practice to master. Routinely, we start students off by learning to dissect and mount third instar eye discs12, which are much easier to work with. This practice helps to develop an appropriate dissection position of the arms, hands and fingers13 so that manipulation of the forceps under the dissecting microscope is stable, easy and experienced. In essence, the practice period shou...

果蝇 ：发育和细胞生物学领域已经通过模式生物受到很大影响。在该模型中，眼睛光盘的研究已经作出了很大贡献有关知识信令，细胞生物学等领域。已故第三龄幼虫眼盘已被广泛研究，并是一个功能强大的模型利用，因为它给出了一系列发育阶段的快照，每个都有其自己的独特信号传导分子和过程，如在整个眼睛光盘的形态发生沟前进8。然而，有必要进一步扩大我们的发展过程的认识到发展的蛹阶段。虽然有研究蛹眼盘3-7，我们的知识不接近已经在三龄眼睛光盘已经完成的工作的广度。这是由于，在某种程度上，在解剖蛹眼睛光盘的更大的困难。因此，一个呈现的解剖适当的方法可以在这方面大大拓展研究。 虽然有在蛹眼睛光盘的发展阶段，很容易解剖，特别是围绕中期蛹期，其他时间段都更有挑战性解剖。该协议代表了一种方法解剖，可普遍用于所有蛹的发育时限蛹的眼睛光盘。这个协议可以用来作为一种替代另一种协议9，其示出了一种更简单和更快速的方法，从midpupal时间点解剖眼光盘。该协议最初拍摄，并在功能基因组学（URCFG）在蛹眼部解剖技术培训10,11先进的本科生在加州大学洛杉矶分校本科生研究协会开发的。许多本科学生能够利用这种视频和方法，了解这一具有挑战性的技术。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Phosphate-buffered saline (PBS, pH 7.4)</td>
			<td></td>
			<td></td>
			<td>80g NaCl, 2g&#160; KCl, 14.4g Na2HPO4, 2.4g KH2PO4, Bring volume to 1 l, adjust the pH to 7.4, autoclave or filter sterilize, dilute to 1X PBS with autoclaved ddH2O before using.</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Promega</td>
			<td>H5142</td>
			<td>Caution: Irritant! Wear gloves.</td>
		</tr>
		<tr>
			<td>0.3% PBT</td>
			<td></td>
			<td></td>
			<td>1.5 ml of Triton X-100, 500 ml 1X PBS.</td>
		</tr>
		<tr>
			<td>37% formaldehyde solution</td>
			<td>Fisher Scientific</td>
			<td>F75P1GAL</td>
			<td>Caution: Toxic, probable human carcinogen! Wear gloves.&#160;</td>
		</tr>
		<tr>
			<td>Fix Solution</td>
			<td></td>
			<td></td>
			<td>(&#8776;4% Formaldehyde in PBS) 50 &#956;l of 37% Formaldehyde, 450 &#956;l 1XPBS, make fresh before use</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Rockland antibodies &#38; assays</td>
			<td>B304</td>
			<td>Aliquot in 1 ml volumes and store at -80C</td>
		</tr>
		<tr>
			<td>Block Solution</td>
			<td></td>
			<td></td>
			<td>10% NGS in PBT.&#160; This can be made and stored at 4 &#176;C for a few days prior to use.</td>
		</tr>
		<tr>
			<td>DAPI stock solution</td>
			<td>Life Technologies</td>
			<td>D3571</td>
			<td>For coutnerstaining nuclei. Prepare a 1 mg/ml solution with ddH2O.</td>
		</tr>
		<tr>
			<td>VectaShield Mounting Medium</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>For mounting. Prepare 70% dilution with ddH2O.</td>
		</tr>
		<tr>
			<td colspan="4">Equipment</td>
		</tr>
		<tr>
			<td>Nutating mixer</td>
			<td>VWR</td>
			<td>82007-202</td>
			<td>Used to rock tissue in 3 well glass dish</td>
		</tr>
		<tr>
			<td>SylGard 182 Silicone Elastomer Kit</td>
			<td>Krayden</td>
			<td>NC9897184</td>
			<td>Used to make silicone dissection dish</td>
		</tr>
		<tr>
			<td>Silicone dissecting dish</td>
			<td></td>
			<td></td>
			<td>Mix Sylgard elastomer kit (above) according to directions gently (to avoid bubbles). Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37 &#176;C incubator.</td>
		</tr>
		<tr>
			<td>3 well glass dish</td>
			<td>Corning</td>
			<td>7220-85</td>
			<td>The 3 well variety of these are no longer available, this is the 9 well product.</td>
		</tr>
		<tr>
			<td>72 well microwell minitray</td>
			<td>Nunc</td>
			<td>438733</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp forceps (Dumont #55)</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td><a name="RANGE!A19">Vannas-type Micro Scissors, Straight, 5mm blade</a></td>
			<td>Ted Pella</td>
			<td>1346</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Borosilicate glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100-4</td>
			<td>Pull with needle puller to make fine point tip that allows a small stream of PBS to flow.</td>
		</tr>
		<tr>
			<td>Disposable Transfer Pipets, Fine Tip</td>
			<td>Samco Scientific</td>
			<td>231</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Tubing dimensions given are inner diameter (ID) x outer diameter (OD) x wall thickness in inches</td>
		</tr>
		<tr>
			<td>PVC tubing (1/8 x 3/16 x 1/32)</td>
			<td>Nalgene</td>
			<td>8000-0010</td>
			<td rowspan="3">Use these with pulled needle to assemble the blower tube as shown in Figure 2.</td>
		</tr>
		<tr>
			<td>Tygon Silicone tubing (3/32 x 5/32 x 1/32)</td>
			<td>Saint Gobain Performance Plastics</td>
			<td>ABW00004</td>
		</tr>
		<tr>
			<td>Tygon Silicone tubing (1/32 x 3/32 x 1/32)</td>
			<td>Saint Gobain Performance Plastics</td>
			<td>ABW00001</td>
		</tr>
	</tbody>
</table>

dissection and mounting of drosophila pupal eye discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

因为使用该协议的一个例子，结果示midpupal（42小时APF，在25℃）进行免疫染色用不同抗体眼光盘示于图2。通过使用针对磷酸酪氨酸残基的抗体，该细胞的膜可以是观察到的（ 图2A）。这可以被用来确定网膜细胞的规则排列在蛹眼以下之前midpupal阶段中发生的最终图案化工艺。另一个代表性图像描绘锥体细胞的细胞核可以通过使用抗体对剪切转录因子（ 图2B）来标识。 <...

Watch this Scientific Journal Video about Dissection and Mounting of Drosophila Pupal Eye Discs at JoVE.com

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

解剖和安装的<em&gt;果蝇</em&gt;蛹眼光盘

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

dissection-and-mounting-of-drosophila-pupal-eye-discs

Research

JoVE Journal

Biology

Dissection and Mounting of Drosophila Pupal Eye Discs

12.1K 次观看.  University of California at Los Angeles (UCLA). The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

视频: Dissection and Mounting of Drosophila Pupal Eye Discs

Dissection of Drosophila Ovaries

剖析果蝇卵巢

科研

生物学

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

从3龄果蝇幼虫的成虫光盘夹层

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 ...

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 ...

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 ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖和免疫染色成虫盘的<em&gt;果蝇</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

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

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

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 ...

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology of the fish branchial skeleton is matched to a species' diet. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a model system to study the genetic and developmental basis of evolved differences in a variety of traits. Marine populations of sticklebacks have repeatedly colonized countless new freshwater lakes and creeks. Adaptation to the new diet in these freshwater environments likely underlies a series of craniofacial changes that have evolved repeatedly in independently derived freshwater populations. These include three major patterning changes to the branchial skeleton: reductions in the number and length of gill raker bones, increases in pharyngeal tooth number, and increased branchial bone lengths. Here we describe a detailed protocol to dissect and flat-mount the internal branchial skeleton in threespine stickleback fish. Dissection of the entire three-dimensional branchial skeleton and mounting it flat into a largely two-dimensional prep allows for the easy visualization and quantification of branchial skeleton morphology. This dissection method is inexpensive, fast, relatively easy, and applicable to a wide variety of fish species. In sticklebacks, this efficient method allows the quantification of skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning.

The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

解剖和三刺鳃骨架的平面安装

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology ...

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Protocols for staging pupal periods and measurement of wing pigmentation of Drosophila guttifera are described. Staging and quantification of pigmentation provide a solid basis for studying developmental mechanisms of adult traits and enable interspecific comparison of trait development.

果蝇 guttifera的分期蛹期和机翼色素沉着的测量方法

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...