Name: imaging dpp release from a drosophila wing disc
Uploaded: 2019-10-30T15:00:00
Description: Watch this Scientific Journal Video about Imaging Dpp Release from a Drosophila Wing Disc at JoVE.com

我们要感谢萨拉拉·普拉丹博士为这一议定书的早期版本开展工作。我们要感谢 NSF-IOS 1354282 在开发此协议时提供的资金。我们要感谢NIH-NIDCR RO1DE025311目前为实验室提供资金。

1. 收集鸡蛋以生成幼虫进行解剖
<ol><li>交叉 30-40 处女 Dpp-GAL4/TM6 Tb Hu 飞到 10-15 男性 Sp/CyO-GFP;UAS-Dpp-GFP/TM6 Tb Hu. 注：只要平衡器具有幼虫标记，允许在幼虫阶段选择适当的后代，可以使用任何包含 Dpp-GAL4 和 UAS-Dpp-GFP 的基因型。</li>
	<li>要收集鸡蛋，将交叉的苍蝇翻转到新鲜的食物小瓶中，让它们放3⁄4小时，然后从小瓶中取出它们。为了鼓励产卵，在将苍蝇引入小瓶之前，可以在食...

<ol>
	<li>Ferguson, E. L., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decapentaplegic+acts+as+a+morphogen+to+organize+dorsal-ventral+pattern+in+the+Drosophila+embryo.">Decapentaplegic acts as a morphogen to organize dorsal-ventral pattern in the Drosophila embryo.</a> Cell. 71 (3), 451-461 (1992).</li><li>Ferguson, E. L., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+enhancement+and+repression+of+the+activity+of+the+TGF-beta+family+member,+decapentaplegic,+is+necessary+for+dorsal-ventral+pattern+formation+in+the+Drosophila+embryo.">Localized enhancement and repression of the activity of the TGF-beta family member, decapentaplegic, is necessary for dorsal-ventral pattern formation in the Drosophila embryo.</a> Development. 114 (3), 583-597 (1992).</li><li>Wharton, K. A., Ray, R. P., Gelbart, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+activity+gradient+of+decapentaplegic+is+necessary+for+the+specification+of+dorsal+pattern+elements+in+the+Drosophila+embryo.">An activity gradient of decapentaplegic is necessary for the specification of dorsal pattern elements in the Drosophila embryo.</a> Development. 117 (2), 807-822 (1993).</li><li>Raftery, L. A., Twombly, V., Wharton, K., Gelbart, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screens+to+identify+elements+of+the+decapentaplegic+signaling+pathway+in+Drosophila.">Genetic screens to identify elements of the decapentaplegic signaling pathway in Drosophila.</a> Genetics. 139 (1), 241-254 (1995).</li><li>Raftery, L. A., Sanicola, M., Blackman, R. K., Gelbart, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+of+decapentaplegic+and+engrailed+expression+in+Drosophila+imaginal+disks:+do+these+genes+mark+the+anterior-posterior+compartment+boundary.">The relationship of decapentaplegic and engrailed expression in Drosophila imaginal disks: do these genes mark the anterior-posterior compartment boundary.</a> Development. 113 (1), 27-33 (1991).</li><li>Blackman, R. K., Sanicola, M., Raftery, L. A., Gillevet, T., Gelbart, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extensive+3'+cis-regulatory+region+directs+the+imaginal+disk+expression+of+decapentaplegic,+a+member+of+the+TGF-beta+family+in+Drosophila.">An extensive 3' cis-regulatory region directs the imaginal disk expression of decapentaplegic, a member of the TGF-beta family in Drosophila.</a> Development. 111 (3), 657-666 (1991).</li><li>de Celis, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+function+of+decapentaplegic+and+thick+veins+during+the+differentiation+of+the+veins+in+the+Drosophila+wing.">Expression and function of decapentaplegic and thick veins during the differentiation of the veins in the Drosophila wing.</a> Development. 124 (5), 1007-1018 (1997).</li><li>De Celis, J. F. <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+wing:+The+development+of+the+veins.">Pattern formation in the Drosophila wing: The development of the veins.</a> Bioessays. 25 (5), 443-451 (2003).</li><li>Letsou, A., 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=Drosophila+Dpp+signaling+is+mediated+by+the+punt+gene+product:+a+dual+ligand-binding+type+II+receptor+of+the+TGF+beta+receptor+family.">Drosophila Dpp signaling is mediated by the punt gene product: a dual ligand-binding type II receptor of the TGF beta receptor family.</a> Cell. 80 (6), 899-908 (1995).</li><li>Nellen, D., Affolter, M., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+serine/threonine+kinases+implicated+in+the+control+of+Drosophila+body+pattern+by+decapentaplegic.">Receptor serine/threonine kinases implicated in the control of Drosophila body pattern by decapentaplegic.</a> Cell. 78 (2), 225-237 (1994).</li><li>Raftery, L. A., Sutherland, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+family+signal+transduction+in+Drosophila+development:+from+Mad+to+Smads.">TGF-beta family signal transduction in Drosophila development: from Mad to Smads.</a> Developmental Biology. 210 (2), 251-268 (1999).</li><li>Dahal, G. R., Pradhan, S. J., Bates, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inwardly+rectifying+potassium+channels+regulate+Dpp+release+in+the+Drosophila+wing+disc.">Inwardly rectifying potassium channels regulate Dpp release in the Drosophila wing disc.</a> Development. 144 (15), 2771-2783 (2017).</li><li>Dahal, G. R., 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=An+inwardly+rectifying+K++channel+is+required+for+patterning.">An inwardly rectifying K+ channel is required for patterning.</a> Development. 139 (19), 3653-3664 (2012).</li><li>George, L. F., 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=Ion+Channel+Contributions+to+Wing+Development+in+Drosophila+melanogaster.">Ion Channel Contributions to Wing Development in Drosophila melanogaster.</a> G3. 9 (4), 999-1008 (2019).</li><li>Huang, H., Liu, S., Kornberg, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+signaling+at+cytoneme+synapses.">Glutamate signaling at cytoneme synapses.</a> Science. 363 (6430), 948-955 (2019).</li><li>Teleman, A. A., Cohen, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dpp+gradient+formation+in+the+Drosophila+wing+imaginal+disc.">Dpp gradient formation in the Drosophila wing imaginal disc.</a> Cell. 103 (6), 971-980 (2000).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394 (6689), 192-195 (1998).</li><li>Dahal, G. R., Pradhan, S. J., Bates, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inwardly+rectifying+potassium+channels+influence+Drosophila+wing+morphogenesis+by+regulating+Dpp+release.">Inwardly rectifying potassium channels influence Drosophila wing morphogenesis by regulating Dpp release.</a> Development. 144 (15), 2771-2783 (2017).</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><li>Hazegh, K. E., Reis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Buoyancy-based+Method+of+Determining+Fat+Levels+in+Drosophila.">A Buoyancy-based Method of Determining Fat Levels in Drosophila.</a> Journal of Visualized Experiments. (117), e54744 (2016).</li><li>Feng, Y., Ueda, A., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> Journal of Neurogenetics. 18 (2), 377-402 (2004).</li><li>Hsiung, F., Ramirez-Weber, F. A., Iwaki, D. D., Kornberg, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+Drosophila+wing+imaginal+disc+cytonemes+on+Decapentaplegic.">Dependence of Drosophila wing imaginal disc cytonemes on Decapentaplegic.</a> Nature. 437 (7058), 560-563 (2005).</li><li>Roy, S., Hsiung, F., Kornberg, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+Drosophila+cytonemes+for+distinct+signaling+pathways.">Specificity of Drosophila cytonemes for distinct signaling pathways.</a> Science. 332 (6027), 354-358 (2011).</li><li>Kornberg, T. B., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytonemes+as+specialized+signaling+filopodia.">Cytonemes as specialized signaling filopodia.</a> Development. 141 (4), 729-736 (2014).</li><li>Roy, S., Huang, H., Liu, S., Kornberg, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoneme-mediated+contact-dependent+transport+of+the+Drosophila+decapentaplegic+signaling+protein.">Cytoneme-mediated contact-dependent transport of the Drosophila decapentaplegic signaling protein.</a> Science. 343 (6173), 1244624 (2014).</li><li>Kornberg, T. B., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Communicating+by+touch--neurons+are+not+alone.">Communicating by touch--neurons are not alone.</a> Trends in Cell Biology. 24 (6), 370-376 (2014).</li></ol>

公诉人等BSP在结合复杂的膜结合受体，在邻近或显然是遥远的细胞中引起细胞内信号的级联时，会产生显著的影响。ThomasKornberg博士的实验室已经表明，产生Dpp信号接触细胞的细胞使用基于活性素的瘦丝波迪亚状结构（称为cytonemes15、24、25）接收信号。这些数据表明，这种背景下的发育细胞-细胞通信可能类似于神经元突<sup clas...

骨形态遗传蛋白（BMPs）对于早期胚胎生成和成人结构模式形成至关重要。BBM 被生成和分泌，以影响响应细胞中生长和细胞分化所需的靶基因的转录。十进制（Dpp）是BMP4的果蝇同源体，对胚胎和成人结构的发展，如翼1，2，3，4是重要的。几个团体关注民进党在图案化成人飞翼的角色，因为1）翅膀由两张透明的上皮片组成，其图案一致，易于评估;2）翼盘也相当平坦，可在幼虫外培养，且易于成像和量化图案差异;3）翼型发展对民进党很敏感，因此路径上的小扰动会影响翼形图案。
Dpp是在位于翼盘5、6、7、8的前/后边界的细胞中产生的。Dpp绑定到1型和2型丝氨酸/色氨酸激酶受体9，10的复合体。在Dpp结合时，2型受体磷化1型受体，然后磷化母亲对Dpp（Mad），一个Smad 1/5/8同源。磷酸化SMAD招募了额外的共同Smad（美地），它使它进入细胞核，在那里它调节目标基因，导致下游效应，如增殖或分化4，11。
最近，贝茨实验室显示，在翼盘内不当释放Dpp会导致狂磷化减少，目标基因表达减少，以及机翼图案缺陷12、13。几个孔通道影响果蝇翼和相关结构的发展14，15。这些子通道也可能参与民进党的释放。在确定变形原释放的机制时，必须采用一种方法来可视化释放事件。
奥雷利奥·泰尔曼博士和斯蒂芬·科恩博士发明了一种Dpp-GFP融合蛋白，能够挽救Dpp的损失，这意味着它在生物学上是活跃的，并且以与生物学相关的方式释放16。在这里，我们描述我们如何可视化 Dpp 发布事件使用此 Dpp-GFP。这种融合蛋白是特别有用的，因为GFP是pH敏感，以至于当它是在酸性囊泡，荧光是淬火17。因此，当标有GFP的蛋白质从囊泡释放到更中性的细胞外环境中时，GFP荧光强度增加17。我们利用GFP的pH敏感性来确定Dpp-GFP是否存在于酸性囊泡中。我们图像翼盘表达Dpp-GFP之前和之后添加氯化铵，其中中和细胞内腔囊泡18。我们发现，在添加氯化铵后，普克塔的荧光显著增加，这表明在添加氯化铵18之前，细胞内Dpp-GFP是淬火的。我们得出结论，细胞内Dpp-GFP驻留在酸性膜结合的隔间，如囊泡，并在添加氯化铵后解脱，以中和细胞内腔内18的pH值。这使得Dpp-GFP的实时成像成为一种有用的技术，可以可视化Dpp在果蝇翼盘中的动态，因为它从酸性隔间释放到细胞外环境中。
在这里，我们描述了我们使用 Dpp-GFP 可视化 Dpp 发布事件的方法。Dpp-GFP可以用UAS-GAL4系统19在果蝇翼盘的本土模式来表达。这是用来确定Irk频道影响Dpp版本18的方法。我们通过实时映像 z 堆栈验证了该方法。如果我们在一个焦点平面上获得，我们看不到Dpp-GFP puncta在时间序列的焦点平面内移动。如果我们在 z 堆栈中成像，我们也看不到 Dpp-GFP 的庞塔移动。我们得出结论，使用这种方法看到的Dpp-GFP puncta是释放事件，而不是细胞内囊泡的运动。这种民进党-GFP的实时成像方法，有可能用于测试民进党释放的其他假定修饰符对民进党动态的影响，也可以修改，以观察其他配体的动态

<table>
	<tbody>
		<tr>
			<td>Baker&#39;s yeast</td>
			<td>Red Star</td>
			<td>&#160;</td>
			<td>&#160;</td>
		</tr>
		<tr>
			<td>CaCl2 dyhydrate</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>VWR</td>
			<td>484-457</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila Agar Type II</td>
			<td>Apex</td>
			<td>66-104</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila melanogaster: Dpp-GAL4/TM6 Tb Hu</td>
			<td></td>
			<td></td>
			<td>This stock will soon be made available at Bloomington Drosophila Stock Center</td>
		</tr>
		<tr>
			<td>Drosophila melanogaster: Sp/CyO-GFP; UAS-Dpp-GFP/TM6 Tb Hu</td>
			<td></td>
			<td></td>
			<td>This stock will soon be made available at Bloomington Drosophila Stock Center</td>
		</tr>
		<tr>
			<td>Dumont Tweezers #5</td>
			<td>World Precision Instruments</td>
			<td>500233</td>
			<td>Forceps for dissecting</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>AC193780010</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Corn Syrup</td>
			<td>Karo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Malt Extract</td>
			<td>Breiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher Scientific</td>
			<td>AC223210010</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Sigma Aldrich</td>
			<td>S8400</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>RPI</td>
			<td>S22060-1000.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>Electron Micsroscopy Sciences</td>
			<td>72180</td>
			<td></td>
		</tr>
		<tr>
			<td>Propionic Acid</td>
			<td>VWR</td>
			<td>U330-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Soy Flour</td>
			<td>ADM Specialty Ingredients</td>
			<td>062-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S5-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S512</td>
			<td></td>
		</tr>
		<tr>
			<td>Tegosept</td>
			<td>Genesee Scientific</td>
			<td>20-259</td>
			<td></td>
		</tr>
		<tr>
			<td>Trehalose dyhydrate</td>
			<td>Chem-Impex International, Inc.</td>
			<td>00766</td>
			<td></td>
		</tr>
		<tr>
			<td>Yellow Corn Meal</td>
			<td>Quaker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM 780 confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Microscope for live imaging</td>
		</tr>
		<tr>
			<td>Zeiss SteREO Discovery.V8 microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Microscope for dissections</td>
		</tr>
	</tbody>
</table>

imaging dpp release from a drosophila wing disc

转化的生长因子-β（TGF-β）超级家族对于多细胞生物中早期胚胎模式的形成和成人结构的发展至关重要。TGF-+超级家族包括TGF-α、骨形态遗传蛋白（BMPs）、活动因子、生长和分化因子以及诺达尔。人们早就知道，暴露于细胞的配体量对它的影响很重要。据认为，远距离浓度梯度建立了胚胎模式。然而，最近很明显，接触这些配体的时机对于它们的下游转录结果也很重要。TGF-+超级家族配体在从产生它的细胞中释放出来之前不能有发育后果。直到最近，还很难确定这些配体何时从细胞中释放出来。在这里，我们展示如何测量一种称为十苯酚（Dpp）的果蝇BMP从翼子板或翼盘的细胞中释放。此方法可以修改为其他系统或信令配体。

图 2显示了该协议的代表性实时映像结果。当协议成功时，Dpp-GFP可以被看作是翼盘中心的条纹，在Dpp-GFP区域中，核可以作为非荧光圆可见（图2）。Dpp-GFP释放是可见的荧光双发，出现和消失。我们观察到Dpp-GFP荧光在细胞体中出现和消失，远离细胞体。民进党信号依赖于基于行为素的菲洛波迪亚状结构，称为cytonemes，远离细胞体22，23。</sup...

Watch this Scientific Journal Video about Imaging Dpp Release from a Drosophila Wing Disc at JoVE.com

Imaging Dpp Release from a Drosophila Wing Disc

接触配体的时机可能会影响其发育后果。在这里，我们展示如何图像释放的果蝇骨形态遗传蛋白（BMP），称为Dpp从翅膀盘的细胞。

成像 Dpp 释放从果蝇翼光盘

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in ...

DPP 通过结合受体和在细胞内发送信号影响转录，影响细胞命运。如果 DPP 产生但未释放，则无法访问受体，因此无法执行其角色。这种技术使得能够可视化D DPP释放，使研究人员能够检查哪些遗传和环境因素改变了D DPP释放动态。BMP信号通路从苍蝇到人类都得到保护，因此我们预计，决定 DPP 释放的机制也将与人类发展和再生相关。首先穿越 30 到 40 处女女性 Dpp GAL4 苍蝇与 10 到 15 雄 UAS DPP - GFP 苍蝇。在新鲜小瓶食物中加入少量酵母酱，鼓励产卵。要收集鸡蛋，翻转交叉苍蝇到小瓶，并允许他们躺在三到四个小时，然后删除他们。将鸡蛋收集瓶保持在25摄氏度左右144小时，直到幼虫处于第三个星形阶段。然后选择适当的幼虫进行解剖。首先，选择非结核的幼虫，这是正常长度，而不是短和脂肪。要选择表达幼虫的DP-GFP，请在荧光立体显微镜下观察它们。所有非结核幼虫都会有荧光，但GP仅限于翼盘，在DP-GFP幼虫上不太亮。根据手稿指示准备经过修改的 HL3 介质，使翼盘保持活成像状态。使用 HEPES 将 pH 调整为 7.1，然后通过 0.22 微米过滤器进行过滤。将两块无色双面胶带宽度放在显微镜幻灯片上，在显微镜幻灯片之间留下大约 5 毫米的空间，并确保胶带的末端与幻灯片的边缘齐平。使用平边将胶带牢固地压在幻灯片上。在两块磁带之间添加 25 个经过修改的 HL3 介质的微升。当翼盘安装在介质中时，胶带片将防止其被盖玻片压碎。要解剖幼虫，请将幼虫放在经过修改的HL3介质中，用两对钳子轻轻撕成两半。丢弃后半部分，然后用一对钳子抓住前半部分的中间，然后用另一对将嘴钩推回幼虫，直到它完全倒置。寻找气管两个较深颜色的主要分支的尖锐弯曲，这些根分支沿着幼虫的两侧运行。翼盘直接位于下方。轻轻取出光盘，并将其放在准备好的幻灯片上的 HL3 介质中，确保没有气管碎片仍然附着在光盘上。排列翼盘，使翼袋的四边朝上，圆盘平躺。用盖玻片盖住机翼，用指甲油密封。抛光干燥后，立即进行成像。使用共和激光扫描显微镜使用 40 倍油目标和 488 纳米激光成像安装的翼盘。以两赫兹的速度收集图像两分钟。为了获得最佳效果，请设置足够强大的激光功率，以获得 DPP 释放细胞的清晰图像，以避免过度漂白。收集图像作为时间序列，并导出为AVI文件，以获得 DPP 发布的视频。当协议成功时，DPP-GFP 会以条纹出现，在翼盘的中心，核以非荧光圆可见。DPP-GFP 释放是可见的荧光朋塔，在细胞体中和远离细胞体中出现和消失。独特的pucta可能是在细胞或在细胞键突触。安装翼盘，以便从四边一开始进行成像。如果翼盘从反面成像，DPP-GFP荧光将出现焦点，分辨率差。当GP不与民进党融合时，在细胞体之外没有观察到任何庞塔。此控件表明 DPP-GFP 的行为与 GFP 本身的行为不同。此外，当不表达 DPP-GFP 时，不会出现荧光朋塔。在此过程期间，翼盘必须安装在上侧。如果翼盘方向不当，DPP-GFP 将出现焦点。此方法可用于研究其他发育信号配体（如无翼或刺猪）的释放。我们可以探讨可能影响民进党释放的所有不同的环境和遗传因素。此外，我们可以调整此方法来研究其他细胞类型的BMP释放。

Research

JoVE Journal

Developmental Biology

Imaging Dpp Release from a Drosophila Wing Disc

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 ...

科研

发育生物学

Upright Imaging of Drosophila Egg Chambers

Upright Imaging of Drosophila Egg Chambers

直立成像<em&gt;果蝇</em&gt;蛋钱伯斯

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

Imaging Calcium in Drosophila at Egg Activation

Imaging Calcium in Drosophila at Egg Activation

在成像钙<em&gt;果蝇</em&gt;在蛋激活

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

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 ...

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 ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

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 ...

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

一种直接而简单的方法，用于评估从胚胎到成人的果蝇黑色素的生存能力

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

成像核内作用棒在活热应力 果蝇 胚胎

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

使用微计算断层扫描的 果蝇黑色素加斯特 全动物成像

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

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, ...