Name: live-cell imaging of drosophila melanogaster third instar larval brains
Uploaded: 2023-06-23T12:40:00.000+00:00
Duration: 7 min 6 s
Description: Watch this Scientific Journal Video about Investigating Asymmetric Cell Division Dynamics: A Protocol for Live-Imaging of Drosophila Larval Brain Explants at JoVE.com

这项研究得到了R35GM148160（C.C.）和美国国立卫生研究院（NIH）培训补助金T32 GM007270（R.C.S）的支持。

注意： 图1 显示了进行本研究所需的材料。
1. 实验的注意事项和准备
<ol><li>防止幼虫过度拥挤。 注意：外植体幼虫大脑的质量与解剖前幼虫的健康和质量直接相关。因过度拥挤而营养不良的幼虫通常会产生质量较低的大脑30。<ol><li>确保每个餐盖培养皿的幼虫不超过20-30只，以避免营养不良。这些示例如图 2 所示。</li>
	</ol></li>
	<li>使用前过滤并等分施耐德培养基。<ol><li>对于每次解剖，通过用1%牛生长血清（BGS）补充等分的施耐德昆虫培养基来制备新鲜的成像和解剖培养基。5 mL 的解剖和成像介质体积通常足以进行成像实验。</li>
		<li>使用前将补充的培养基加热至室温 （RT）。</li>
	</ol></li>
	<li>考虑要收集的短片的长度，并用它来考虑成像介质的补充、安装方法和显微镜的采集设置。 注意：在最佳条件下，仅补充BGS的大脑中的NB将强有力地分裂3小时以上。<ol><li>通过在成像介质中添加幼虫脂肪体组织来补充成像培养基，以支持在进行需要更长电影的实验时超过4小时的分裂。 注意：脂肪体分泌支持NB增殖的有丝分裂原31，来自10个幼虫的整个脂肪体足以支持4到5个大脑。此外，用膜结合载玻片成像的样品已被证明分裂超过10小时13,32，而用多孔载玻片成像的样品通常分裂的频率较低（未发表的观察结果）。</li>
		<li>或者，为较长的电影实现更复杂的成像介质，如前所述33.通过调整曝光时间、激光功率和采样频率来最大程度地减少光损伤，以获得最佳效果。</li>
	</ol></li>
</ol>2. 幼虫分期和收集（图2）
<ol><li>将1-5日龄的雌性处女苍蝇与1-7日龄的成年雄性苍蝇杂交，以产生具有所需基因型的后代。为了获得最佳产量，将 10-15 个女性处女与 5-10 个男性杂交。将这些苍蝇放入带有餐盖的苍蝇笼中（图2A-C），并在25°C下孵育。</li>
	<li>每天交换餐盖。这可以防止餐盖被幼虫过度拥挤，从而降低解剖大脑的质量。</li>
	<li>如果餐盖被幼虫明显覆盖（即>30），请将该餐盖分成两半，并用同样切成两半的新鲜餐盖代替一半。或者，更频繁地交换餐盖（即每 12 小时而不是每 24 小时）。餐帽过多的示例如图2E，F所示。</li>
	<li>将餐盖与幼虫在25°C孵育，直到幼虫达到所需年龄。</li>
</ol>3.幼虫脂肪体解剖（图3）
注意：本协议描述了使用3孔解剖皿的解剖。
<ol><li>将 ~400 μL 解剖和成像培养基移液到 3 孔解剖皿的每个孔中。</li>
	<li>用解剖钳轻轻握住十只 72-96 小时龄喂养良好的野生型幼虫，并将它们浸入和浸出最底部孔中的解剖溶液，直到所有食物颗粒都被洗掉。冲洗后，将干净的幼虫移到中间孔中。</li>
	<li>使用一组镊子，用嘴钩握住幼虫。用另一组镊子，破坏幼虫角质层的一侧。</li>
	<li>这种破裂会导致脂肪体从幼虫中溢出。脂肪体是灰白色和半透明的，将具有格子状结构（图3I）。肥胖的身体也倾向于粘在自己和解剖镊子上。一旦确定，从每个幼虫中收集尽可能多的脂肪体，并用镊子将其转移到带有 400 μL RT 解剖培养基的最顶层孔中。</li>
</ol>4.幼虫脑解剖（图3）
<ol><li>如上所述在解剖和成像介质中清洗实验幼虫，以清除它们的食物残渣。为获得最佳效果，请避免将未解剖的幼虫存放在解剖溶液中。这将导致幼虫“淹死”，并对解剖大脑的质量产生负面影响。</li>
	<li>使用一组镊子，用嘴钩握住幼虫。使用另一组镊子，从后侧轻轻切割/撕下大约 1/3 的幼虫（图 3A）。这将导致消化道，脂肪体，结缔组织和神经系统的元素从幼虫破裂的一侧“爆发”出来（图3B）。</li>
	<li>使用一组镊子，用嘴钩握住幼虫。用另一组镊子，轻轻地将角质层刷向口钩，同时用镊子握住口钩向内“推动”，直到整个幼虫翻过来。这种运动类似于将袜子“从内到外”转动（图3C，D）。</li>
	<li>将幼虫倒置，使中枢神经系统和其他组织朝外，同时仍与角质层相连。在此步骤中，找到中枢神经系统（CNS）以避免意外移除。使用镊子轻轻去除所有非CNS组织，仅留下附着在角质层上的CNS和大脑（图3E）。</li>
	<li>大脑将通过轴突连接 附着 在角质层上。使用显微解剖剪刀，切断这些轴突连接，将大脑从角质层释放出来。为此，首先轻轻切开脑叶下方（图3F）。重复腹侧神经索下的连接。 注意：如果没有显微切割剪刀，则可以使用镊子完成此步骤。使用镊子时要特别小心，以确保脑组织在从角质层取出时不会过度拉伸，因为机械应力会对大脑健康产生负面影响。</li>
	<li>将解剖的大脑转移到带有解剖和成像介质的孔中。对于超过3小时的成像实验，如上所述使用补充脂肪体的解剖和成像介质。分批解剖幼虫，以保持解剖时间在20分钟以下。</li>
</ol>5. 安装和成像（图4）
<ol><li>对于使用膜结合玻片34成像：<ol><li>在解剖盘的最后一口井中收集解剖的大脑和孤立的脂肪体。</li>
		<li>通过在载玻片背面放置透气膜来组装载玻片的一半，然后将分体环压入中心，将其固定到位（图4A-C）。</li>
		<li>使用 200 μL 微量移液器，将 ~130-140 μL 解剖和成像介质中的多达 10 个解剖大脑和尽可能多的脂肪体（见上文）转移到膜上。确保将培养基与样品沉积在透气膜的中心（图4D，E）。</li>
		<li>针对要成像的NB群体和正在使用的显微镜类型定向大脑（图4E）。将样品放置在尽可能靠近显微镜物镜的位置。例如，要对中央脑叶中的NB进行成像，请调整大脑的方向，使脑叶最接近目标（图4H）。</li>
		<li>一旦大脑定向，轻轻地将玻璃盖玻片放在膜上的溶液顶部。这将导致含有大脑和脂肪体的溶液扩散到整个膜上（图4F）。</li>
		<li>通过将实验室组织靠近盖玻片边缘来吸干过量的溶液。当大脑接触盖玻片而不被压扁时，可以达到最佳溶液量。如果在此步骤中需要重新定向，请小心地移动盖玻片以移动大脑。</li>
		<li>用画笔沿着盖玻片的边缘涂抹融化的凡士林来固定盖玻片。让果冻凝固（图4G）。</li>
	</ol></li>
	<li>对于使用多孔成像载玻片成像（图4）：<ol><li>将 400 μL 成像培养基添加到多孔载玻片的孔中（在此处进行的实验中，使用腔室 8 孔微型 [μ] 载玻片; 图4I）。将先前解剖的脂肪体转移到该孔中（见步骤3.4）。</li>
		<li>在孔中心附近的簇中沉积多达10个大脑（图4J）。</li>
		<li>如步骤5.1.4（图4K）中所述，针对要成像的NB群体和正在使用的显微镜类型定向大脑。排列样本，使它们彼此靠近。这将最大限度地减少样品台在样品之间必须移动的距离，从而减少采集过程中的样品漂移。</li>
		<li>一旦大脑在井中定向，让大脑沉淀2-5分钟。这增加了它们在运输/成像过程中的稳定性。在此期间准备显微镜以进行采集。</li>
		<li>用载玻片盖盖住载玻片μ载玻片，然后将其转移到显微镜上。以尽可能低的激光功率和曝光时间开始采集，以最大限度地减少光漂白。</li>
	</ol></li>
</ol>6. 数据处理和管理最佳实践
<ol><li>根据可用的分析软件根据需要处理数据。<ol><li>对于此处显示的示例，使用幻灯片软件将采集的数据保存为幻灯片图像文件 （.sld）。</li>
		<li>要使用 Imaris 文件转换器转换为 Imaris 的专有文件类型 （.ims），请在单独的窗口中打开 Imaris 文件转换器。单击并将.sld文件拖到Imaris文件转换器的“输入”部分。</li>
		<li>确定转换后文件的所需输出位置，然后单击“全部启动”。</li>
		<li>转换后，在Imaris软件中查看和注释数据。 注意：可以使用图像分析的替代方案代替Imaris，例如斐济（https://hpc.nih.gov/apps/Fiji.html），Aivia（https://www.aivia-software.com/），Volocity（https://www.volocity4d.com/）或其他。</li>
	</ol></li>
	<li>保留尽可能多的原始数据，以便妥善保存记录。例如，如果采集软件保存为一种文件格式，但转换为另一种格式进行分析，请保留采集的数据版本。</li>
	<li>对于数据分析，请尽可能多地记录有关样品和采集设置的详细信息。要保留的关键信息包括解剖幼虫的基因型、解剖前幼虫的年龄、饲养它们的餐盖状态、成像期间使用的激光功率、曝光时间、采集时间和时间分辨率。</li>
</ol>7. 细胞周期长度定量示例（图5）
注意：在本例中，对表达极性标记针（Pins：：EGFP16）和微管结合蛋白木星25 （樱桃：：木星13）的幼虫进行成像。随后的分析是使用Imaris软件进行的。
<ol><li>使用所选的图像分析软件打开电影。滚动浏览电影的长度以识别划分的 NB，并标记它们以供将来参考。通过其不同的有丝分裂纺锤体识别分裂的NB（图5C-E）。</li>
	<li>确定参考细胞周期阶段以确定细胞周期长度。在本例中，中期用作参考。</li>
	<li>手动确定连续中期之间的帧数，并将其转换为分钟或小时以确定完成一个细胞周期所需的时间。<ol><li>为此，请采用电影的时间分辨率并将其乘以中期之间的帧数。例如，如果电影的时间分辨率为每 5 分钟一帧，并且在第 13 帧和第 35 帧观察到中期，则这些中期之间的时间为 110 分钟（[35 − 13] × 5）。</li>
	</ol></li>
	<li>使用任何适当的软件绘制数据。此处显示的数据是使用PRISM软件绘制的。</li>
</ol>8. 细胞纺锤对准的量化示例（图5）
注意：在本例中，使用 Imaris 软件执行分析。
<ol><li>在 Imaris 或其他所选软件中打开电影文件。滚动浏览电影的长度以识别划分的 NB，并标记它们以供将来参考。</li>
	<li>使用顶端和基底中心体（用 m表示）确定纺锤体极形成的载体，如下所示： <img alt="Equation 1" src="/files/ftp_upload/65538/65538eq01.jpg" style="margin: auto;"> 其中 Ax、 Ay 和 Az 是顶端中心体的坐标， Bx、 By 和 Bz 是基底中心体的坐标。类似地，分裂向量的轴（用 n表示）由顶端Pins：：EGFP新月形和基底皮层的中点形成： <img alt="Equation 2" src="/files/ftp_upload/65538/65538eq02.jpg" style="margin: auto;"> 其中 Ax、 Ay 和 Az 是 Pins：：EGFP 新月形中点的坐标， Bx、 By 和 Bz 是基底皮层中点的坐标。</li>
	<li>确定向量 m 和 n 的大小： m的星等：<img alt="Equation 3" src="/files/ftp_upload/65538/65538eq03.jpg" style="margin: auto;"> n 的大小：<img alt="Equation 4" src="/files/ftp_upload/65538/65538eq04.jpg" style="margin: auto;"></li>
	<li>确定 m 和 n 的点积（用 k 表示）： <img alt="Equation 5" src="/files/ftp_upload/65538/65538eq05.jpg" style="margin: auto;"></li>
	<li>使用点积 k 和矢量幅度 m 和 n，确定矢量之间的角度： <img alt="Equation 6" src="/files/ftp_upload/65538/65538eq06.jpg" style="margin: auto;"></li>
	<li>在所选软件中绘制数据。此处显示的数据是在 Excel Microsoft 中准备的，并在 PRISM 中可视化。</li>
</ol>

来自 黑腹果蝇 第三龄幼虫的活脑外植体成像

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S., Otte, F., Cabernard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+polarity+regulates+biased+myosin+activity+and+dynamics+during+asymmetric+cell+division+via+Drosophila+rho+kinase+and+protein+kinase+N.">Cell polarity regulates biased myosin activity and dynamics during asymmetric cell division via Drosophila rho kinase and protein kinase N.</a> Developmental Cell. 42 (2), 143-155 (2017).</li><li>Montembault, E., 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=Myosin+efflux+promotes+cell+elongation+to+coordinate+chromosome+segregation+with+cell+cleavage.">Myosin efflux promotes cell elongation to coordinate chromosome segregation with cell cleavage.</a> Nature Communications. 8 (1), 326 (2017).</li><li>Roubinet, C., 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=Spatio-temporally+separated+cortical+flows+and+spindle+geometry+establish+physical+asymmetry+in+fly+neural+stem+cells.">Spatio-temporally separated cortical flows and spindle geometry establish physical asymmetry in fly neural stem cells.</a> Nature Communications. 8 (1), 1383 (2017).</li><li>Januschke, 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=Centrobin+controls+mother-daughter+centriole+asymmetry+in+Drosophila+neuroblasts.">Centrobin controls mother-daughter centriole asymmetry in Drosophila neuroblasts.</a> Nature Cell Biology. 15 (3), 241-248 (2013).</li><li>Januschke, J., Llamazares, S., Reina, J., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+neuroblasts+retain+the+daughter+centrosome.">Drosophila neuroblasts retain the daughter centrosome.</a> Nature Communications. 2 (1), 243 (2011).</li><li>Rebollo, E., 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=Functionally+unequal+centrosomes+drive+spindle+orientation+in+asymmetrically+dividing+Drosophila+neural+stem+cells.">Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells.</a> Developmental Cell. 12 (3), 467-474 (2007).</li><li>Januschke, J., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interphase+microtubule+aster+is+a+determinant+of+asymmetric+division+orientation+in+Drosophila+neuroblasts.">The interphase microtubule aster is a determinant of asymmetric division orientation in Drosophila neuroblasts.</a> The Journal of Cell Biology. 188 (5), 693-706 (2010).</li><li>Rusan, N. M., Peifer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+a+novel+centrosome+cycle+in+asymmetric+cell+division.">A role for a novel centrosome cycle in asymmetric cell division.</a> The Journal of Cell Biology. 177 (1), 13-20 (2007).</li><li>Lerit, D. 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=Interphase+centrosome+organization+by+the+PLP-Cnn+scaffold+is+required+for+centrosome+function.">Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function.</a> Journal of Cell Biology. 210 (1), 79-97 (2015).</li><li>Gallaud, E., 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=Dynamic+centriolar+localization+of+Polo+and+Centrobin+in+early+mitosis+primes+centrosome+asymmetry.">Dynamic centriolar localization of Polo and Centrobin in early mitosis primes centrosome asymmetry.</a> PLoS Biology. 18 (8), e3000762 (2020).</li><li>Ramdas Nair, 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=The+microcephaly-associated+protein+Wdr62/CG7337+is+required+to+maintain+centrosome+asymmetry+in+Drosophila+neuroblasts.">The microcephaly-associated protein Wdr62/CG7337 is required to maintain centrosome asymmetry in Drosophila neuroblasts.</a> Cell Reports. 14 (5), 1100-1113 (2016).</li><li>Singh, P., Nair, A. R., Cabernard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+centriolar+protein+Bld10/Cep135+is+required+to+establish+centrosome+asymmetry+in+Drosophila+neuroblasts.">The centriolar protein Bld10/Cep135 is required to establish centrosome asymmetry in Drosophila neuroblasts.</a> Current Biology. 24 (13), 1548-1555 (2014).</li><li>LaFoya, B., Prehoda, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consumption+of+a+polarized+membrane+reservoir+drives+asymmetric+membrane+expansion+during+the+unequal+divisions+of+neural+stem+cells.">Consumption of a polarized membrane reservoir drives asymmetric membrane expansion during the unequal divisions of neural stem cells.</a> Developmental Cell. 1534 (23), 00159 (2023).</li><li>Sunchu, 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=Asymmetric+chromatin+retention+and+nuclear+envelopes+separate+chromosomes+in+fused+cells+in+vivo.">Asymmetric chromatin retention and nuclear envelopes separate chromosomes in fused cells in vivo.</a> Communications Biology. 5 (1), 953 (2022).</li><li>Oliveira, A. C., Rebelo, A. R., Homem, C. 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=Integrating+animal+development:+How+hormones+and+metabolism+regulate+developmental+transitions+and+brain+formation.">Integrating animal development: How hormones and metabolism regulate developmental transitions and brain formation.</a> Developmental Biology. 475, 256-264 (2021).</li><li>Britton, J. S., Edgar, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+control+of+the+cell+cycle+in+Drosophila:+nutrition+activates+mitotic+and+endoreplicative+cells+by+distinct+mechanisms.">Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms.</a> Development. 125 (11), 2149-2158 (1998).</li><li>Lee, C. -. 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=Drosophila+Aurora-A+kinase+inhibits+neuroblast+self-renewal+by+regulating+aPKC/Numb+cortical+polarity+and+spindle+orientation.">Drosophila Aurora-A kinase inhibits neuroblast self-renewal by regulating aPKC/Numb cortical polarity and spindle orientation.</a> Genes &amp; Development. 20 (24), 3464-3474 (2006).</li><li>Homem, C. C. F., Reichardt, I., Berger, C., Lendl, T., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+live+cell+imaging+and+automated+4D+analysis+of+Drosophila+neuroblast+lineages.">Long-term live cell imaging and automated 4D analysis of Drosophila neuroblast lineages.</a> PLoS ONE. 8 (11), e79588 (2013).</li><li>Cabernard, C., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+neuroblast+lineages+within+intact+larval+brains+in+Drosophila.">Live imaging of neuroblast lineages within intact larval brains in Drosophila.</a> Cold Spring Harbor Protocols. 2013 (10), 970-977 (2013).</li><li>Karpova, N., Bobinnec, Y., Fouix, S., Huitorel, P., Debec, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jupiter,+a+new+Drosophila+protein+associated+with+microtubules.">Jupiter, a new Drosophila protein associated with microtubules.</a> Cell Motility and the Cytoskeleton. 63 (5), 301-312 (2006).</li><li>Loyer, N., Januschke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+last-born+daughter+cell+contributes+to+division+orientation+of+Drosophila+larval+neuroblasts.">The last-born daughter cell contributes to division orientation of Drosophila larval neuroblasts.</a> Nature Communications. 9 (1), 3745 (2018).</li><li>Bostock, M. 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作者没有财务披露需要申报。

该协议概述了一种从 黑腹果蝇 幼虫对活外植体大脑进行成像的方法。这里描述的方案允许在正确的实验条件下观察外植体大脑12-20小时。必须特别考虑样品的制备和所需实验的设计。如上所述，决定解剖组织质量的最关键因素之一是幼虫的健康。为了达到尽可能高的质量，必须确保幼虫在采集前得到良好的喂养。不健康的幼虫最常见的原因是过度拥挤。为了解决这个问题，必须确保通过增...

不对称细胞分裂 （ACD） 是亚细胞成分（如 RNA、蛋白质和细胞器）在子细胞之间分配不均等的过程 1,2。这个过程在干细胞中很常见，干细胞经历ACD以产生具有不同发育命运的子细胞。果蝇NBs不对称地分裂以产生一个NB，保留其干性，以及一个神经节母细胞（GMC）。GMC经历进一步的分裂以产生分化的神经元或神经胶质细胞3。不对称分裂的NBs在三龄幼虫的发育大脑中很丰富，通过显微镜很容易观察到。在第三龄幼虫阶段，每个中枢脑叶中大约有100个NBs3,4,5,6。
不对称细胞分裂是一个高度动态的过程。活细胞成像方案已被用于测量和量化细胞极性7,8,9,10，纺锤体取向11,12,13，肌动肌蛋白皮层14,15,16,17,18，微管和中心体生物学19,20的动力学，21,22,23,24,25,26,27和膜10,28和染色质动力学29。ACD的定性和定量描述依赖于可靠的方法和协议来成像完整活体大脑中的NBs分割。以下协议概述了使用两种不同的安装方法制备，解剖和成像用于体内活细胞成像的第三龄幼虫大脑的方法。这些方法最适合对干细胞分裂以及其他脑细胞分裂的时空动力学感兴趣的研究人员，因为它们允许对细胞事件进行短期和长期观察。此外，这些技术对于该领域的新手来说很容易获得。我们证明了这种方法在表达荧光标记的微管和皮质融合蛋白的幼虫脑中的有效性和适应性。我们还讨论了分析方法和在其他研究中应用的注意事项。

<table><tbody><tr><td>0.22 &amp;micro;m polyethersulfone (PES) Membrane</td><td>Genesee</td><td>25-231</td><td>Vacuum-driven filters</td></tr><tr><td>Agar</td><td>Genesee</td><td>20-248</td><td>granulated agar</td></tr><tr><td>Analytical Computer</td><td>Dell</td><td>NA</td><td>Intel Xeon Gold 5222 CPU with two 3.80 GHz processors running Windows 10 on a 64-bit operating system</td></tr><tr><td>Bovine Growth Serum</td><td>HyClone</td><td>SH30541.02</td><td></td></tr><tr><td>Chambered Imaging Slides</td><td>Ibidi</td><td>80826</td><td></td></tr><tr><td>Confocal Microscope</td><td>Nikon</td><td>NA</td><td></td></tr><tr><td>Custom-machined metal slide</td><td>NA</td><td>NA</td><td>See Cabernard and Doe 2013 (Ref. 34) for specifications</td></tr><tr><td>Dissection Dishes</td><td>Fisher Scientific</td><td>5024343</td><td>3-well porcelain micro spot plate</td></tr><tr><td>Dissection Forceps</td><td>World Precision Instruments</td><td>Dumont #5</td><td></td></tr><tr><td>Dissection Microscope</td><td>Leica</td><td>NA</td><td></td></tr><tr><td>Dissection Scissors</td><td>Fine Science Tools (FST)</td><td>15003-08</td><td></td></tr><tr><td>Embryo collection cage</td><td>Genesee</td><td>59-100</td><td></td></tr><tr><td>Flypad with access to CO2 to anesthetize adult flies</td><td>Genesee</td><td>59-172</td><td></td></tr><tr><td>Gas-permeable membrane</td><td>YSI</td><td>98095</td><td>Gas-permeable membrane</td></tr><tr><td>Glass Cover Slides</td><td>Electron Microscopy Sciences</td><td>72204-03</td><td># 1.5; 22 mm x 40 mm glass coverslips</td></tr><tr><td>Imaris</td><td>Oxford Instruments</td><td>NA</td><td>Alternatives: Fiji, Volocity, Aivia</td></tr><tr><td>Imaris File Converter</td><td>Oxford Instruments</td><td>NA</td><td></td></tr><tr><td>Instant Yeast</td><td>Saf-Instant</td><td>NA</td><td></td></tr><tr><td>Molasses</td><td>Genesee</td><td>62-117</td><td></td></tr><tr><td>Petri dish</td><td>Greiner Bio-One</td><td>628161</td><td>60 mm x 15 mm Petri dish</td></tr><tr><td>Petroleum Jelly</td><td>Vaseline</td><td>NA</td><td></td></tr><tr><td>Schneider&#039;s Insect Medium with L-glutamine and sodium bicarbonate liquid</td><td>Millipore Sigma</td><td>S0146</td><td></td></tr><tr><td>SlideBook acquisition software</td><td>3i</td><td>NA</td><td></td></tr><tr><td>Vacuum-Driven Filtration Unit with a 0.22 &amp;micro;&amp;micro;m PES membrane filter</td><td>Genesee Scientific, GenClone</td><td>25-231</td><td></td></tr></tbody></table>

live-cell imaging of drosophila melanogaster third instar larval brains

果蝇 神经干细胞（神经母细胞，以下简称NBs）经历不对称分裂，再生自我更新的神经母细胞，同时还形成分化神经节母细胞（GMC），该母细胞将经历一次额外的分裂以产生两个神经元或神经胶质细胞。NBs的研究揭示了细胞极性，纺锤体取向，神经干细胞自我更新和分化的分子机制。这些不对称的细胞分裂很容易通过活细胞成像 观察到 ，使幼虫NB非常适合研究活组织中不对称细胞分裂的时空动力学。当在营养补充培养基中正确解剖和成像时，外植体大脑中的NBs强烈分裂12-20小时。前面描述的方法在技术上是困难的，对于那些刚进入该领域的人来说可能具有挑战性。在这里，描述了使用脂肪体补充剂制备、解剖、安装和成像活的三龄幼虫脑外植体的方案。还讨论了潜在的问题，并提供了如何使用这种技术的示例。

中枢脑叶NBs的解剖和成像表达Pins：：EGFP和樱桃：：木星 为了展示这一协议，表达UAS驱动的樱桃：：木星13和内源性标记的Pins：：EGFP16（w; worGal4，UAS-cherry：：jupiter/CyO;引脚：：EGFP / TM6B，Tb）使用多孔成像载玻片使用所述方案成像4小时（图5C，D）。其他数据取自表达UAS驱动的樱桃：：木星13的...

Watch this Scientific Journal Video about Investigating Asymmetric Cell Division Dynamics: A Protocol for Live-Imaging of Drosophila Larval Brain Explants at JoVE.com

作者聚焦：研究不对称细胞分裂动力学：果蝇幼虫脑外植体实时成像的方案 

在这里，我们讨论了一种工作流程，用于准备、解剖、安装和成像来自黑 腹果蝇 第三龄幼虫的活外植体大脑，以观察生理条件下的细胞和亚细胞动力学。

黑腹果蝇第三龄幼虫脑的活细胞成像

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...

我们的研究重点是研究不对称细胞分裂或ACD对干细胞增殖和分化的影响。我们使用果蝇神经干细胞或神经母细胞作为模型来了解不对称细胞分裂对发育和神经发生的机制、法规和影响。人工智能的最新发展在图像量化和分析方面取得了重大进展，这大大推动了生命细胞成像领域的发展。保持长期电影的采样质量是一项长期挑战。虽然观察样品 8 到 10 小时很有见地，但后勤限制，如样品的成像要求和可用的成像工具，使其变得困难。我们的实验方案通过在不影响样品质量的情况下展示长期成像来解决这个问题。我们的技术可以很容易地被新来者采用和应用于该领域。通过一些时间和实践，人们可以制作短期和长期电影，这些电影有可能产生有见地和有意义的数据。

live-cell-imaging-drosophila-melanogaster-third-instar-larval

Research

JoVE Journal

Developmental Biology

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

3.3K 次观看.  University of Washington. 我们的研究重点是研究不对称细胞分裂或ACD对干细胞增殖和分化的影响。我们使用果蝇神经干细胞或神经母细胞作为模型来了解不对称细胞分裂对发育和神经发生的机制、法规和影响。人工智能的最新发展在图像量化和分析方面取得了重大进展，这大大推动了生命细胞成像领域的发展。保持长期电影的采样质量是一项长期挑战。虽然观察样品 8 到 10 小时很有见地，但后...

视频: 作者聚焦：研究不对称细胞分裂动力学：果蝇幼虫脑外植体实时成像的方案 

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

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 ...

科研

发育生物学

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

3幼虫节段性神经的可视化 RD龄果蝇幼虫的筹备工作

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

神经科学

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

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

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

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

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 ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

住在果蝇眼睛成虫光盘胶质细胞迁移成像

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

生物学

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

发展和成人的制备果蝇大脑和视网膜实时成像

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Real-Time Imaging of Germline Stem Cells in Drosophila Ex Vivo Germaria Culture

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs&#39; asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

黑腹果蝇第三龄幼虫脑的活细胞成像

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