Name: live imaging of mouse secondary palate fusion
Uploaded: 2017-07-27T14:00:00.000+00:00
Duration: 6 min 10 s
Description: Watch this Scientific Journal Video about Live Imaging of Mouse Secondary Palate Fusion at JoVE.com

我们感谢道格拉斯·本森先生关于二次上颚成像的初步对话。我们还承认David Castaneda-Castellanos（Leica）和Chris Rieken（蔡司）帮助调整共聚焦显微镜中的成像条件。感谢Lynsey Hamilton（Bitplane）对使用Imaris软件进行定量图像分析的有用建议。这项工作由NIH / NIDCR R01 DE025887资助。

所有动物实验均按照加利福尼亚大学旧金山机构动物护理和使用委员会的协议进行。 活动影像制作准备<ol><li> 液体培养基的制备 <ol><li>向Dulbecco&#39;s Modified Eagle培养基（DMEM）/ F12中加入20％胎牛血清（FBS），2mM L-谷氨酰胺，100U / mL青霉素，100μg/ mL链霉素，200μg/ mL L-抗坏血酸，15mM HEPES媒体。 注意:在成像之前，实时成像介质是新鲜的。这种介质含有酚红，这可能导致长时间的活体成像过程中的光毒性。代替不含酚红的培养基可以改善长期成像。 </li></ol></li><li> 制备低熔点琼脂糖溶液 <ol><li>将350毫克低熔点琼脂糖溶解在10毫升高压灭菌的蒸馏水中以制备3.5％储备溶液。 </li><li>将琼脂糖溶液充分混合，并在70℃在水浴中孵育以完全溶解琼脂糖。 </li><li>在37℃水浴中冷却琼脂糖溶液，然后将其加入到活体成像介质中。 </li><li>将1 mL琼脂糖储备液加入5 mL液体活体成像培养基中，使其达到最终浓度（0.6％）。 </li><li>将培养基用琼脂糖保存在37°C水浴中，以防止过早凝固。 </li></ol></li></ol> 2.准备活体外植体文化<ol><li> 近期附着的E14.5二级腭架的解剖 注意:在我们的经验中，成像融合需要从轻柔的一对腭架开始;这种粘附防止在成像期间防止融合发生的外植体运动。 <ol><li>在1x磷酸缓冲盐水（PBS）中解剖E14.5期胚胎，并选择绿色荧光蛋白（GFP）表达（来自K14-CRE; ROSA26-mTmG flox ）或（红色荧光蛋白）表达RFP（来自Lifeact-mRFPruby ）的阳性胚胎，使用装备有荧光灯的解剖显微镜。将组织转移到没有琼脂糖的预热活体成像培养基中。 </li><li>切割胚胎头并使用两个5号镊子去除上脑区域（ 图 1A-C ）。使用5号镊子握住胚胎，而另外5号镊子切除腭架外的额外组织。 注意:精剪可用于切割胚胎头（第一步），而不是一个5号镊子。然而，两个5号细镊子将能够更好地控制精确切割。 </li><li>没有如图1（D）和图1E损坏腭小心取出下颌骨。为了减少腭裂的机会，保持舌头通过下颌骨的解剖，f通过仔细去除舌头。 </li><li>在去除下颌骨和舌头后，用立体显微镜用荧光检查二尖瓣的口腔侧面，以确认MES中的强报道信号（ 图1 M ）。 </li><li>在去除下颌骨和舌后解剖后部部分，包括后脑和脑干（ 图 1F ）。 </li><li>用粘附的腭架除去两个上颌骨（ 图1 G ）。 </li><li>切割前上​​颌组织保持原发性和次级腭部完整（ 图1 H和I ）。 </li><li>去除露出MES的外植体鼻侧的鼻中隔。 注意:这些以后的解剖步骤需要注意，不要破坏两个辅助腭架之间的接触。去除鼻中隔有助于减少其他区域的背景信号，并减少垂直组织移动，使稳定成像（ 图1 J-L ）。 </li><li>解剖腭架后，再次检查中线记者信号，确保组织之间上皮层无损伤。 </li></ol></li><li> 在活体成像介质的培养皿中设置腭外植体 <ol><li>将活体成像介质放入35毫米玻璃底盘中。 </li><li>将解剖的腭骨架面朝下，尽可能靠近玻璃底部，用倒置共聚焦显微镜进行成像（ 图 N1 ）。 </li><li>将干冰粒放在培养皿附近的导电表面上，以快速降温或将培养皿放置在4℃加速琼脂糖凝固。也可以使用冷板，如用于石蜡包埋的冷板踏歌。 注意:当使用干冰时，需要仔细监测琼脂糖介质的变化以防止冷冻介质。 </li></ol></li></ol>腭内窥镜的共焦延时成像<ol><li> 成像和数据采集 <ol><li>琼脂糖半固化后，将培养皿安装在装有37℃孵育室的倒置共焦显微镜中。使用白光共焦显微镜和细胞观察器旋转盘共聚焦显微镜。 注意:我们使用含有15mM的HEPES无CO 2的气体的修改的媒体。 </li><li>使用注射器将培养皿和盖子之间的果冻放入培养基中以防止培养基蒸发（ 图 N ）。 注意:过夜（O / N）培养发生后，培养皿盖顶部有一些冷凝，但介质的体积不会改变，这表明石油胶可以防止培养基蒸发。 </li><li>使用低放大倍数目标（10x）找到感兴趣的区域，并使用更高放大倍数的目标（20倍物镜，3倍数码变焦或40x物镜与Immersol W）进行延时实时成像。 注意:解剖后的腭骨架不平坦。二级腭是一个弯曲的结构，这使得难以在同一平面上对整个MES进行成像。低倍率（10倍）成像可以允许全腭成像，但40倍的目标将提供更好的图像分辨率，以检查更深的Z位置的细胞细胞运动。中腭区曲率高。与中腭相比，前后腭相对平坦。我们经常朝向二级腭中段前方，以避免曲率最大的区域。 </li><li>使用适当的激光激发（对于GFP为488nm，RFP为532/561nm）扫描多个Z堆叠，每步5μm为16-24小时（ 图2 ）。 注意:为了降低潜在的光毒性，请使用最小的激光功率和曝光时间。特别是在多色成像的情况下尤为重要。使用52nm激光功率的22％用于membraneTomato（mT），30％的495nm激光功率用于膜GFP（mG）和25％的561nm激光功率用于膜RFP。平均曝光时间为550 ms。 </li><li>使用图像分析软件执行细胞跟踪和定量分析。 </li></ol></li></ol>数据图像分析注意:这里使用了Imaris。也可以使用ImageJ或Volocity软件包执行类似的数据分析。 <ol><li> 从图像序列渲染三维（3D）曲面。 注意:本部分可以从使用膜GFP信号生成的影片中生成曲面。 <ol><li>单击编辑|显示显示调整并使用直方图滑块调整信号强度，使膜信号清晰出片的长度（ 图3 的A）。 </li><li>使用图像正确漂白的信号 处理|衰减校正 。调整强度前沿和强度返回值，以使膜信号在整个电影长度内清晰。设置这些值可能需要一些试验和错误。 注意:由于激光穿透受到限制，强度返回（更深的Z位置）值将小于强度前沿（表面Z位置）值。因此，这些值也可以改变为使Z系列中的信号强度正常化。可以调整这两个值以实现漂白校正。衰减校正也称为发射衰减校正19 。 </li><li>为了从膜信号产生表面，选择Surpass |表面 ，选择源通道和阈值类型（绝对强度），并在算法设置下LECT 仅划分兴趣区域 并完成整个图像的处理 ，然后点击向前箭头;继续按向前箭头（使用默认设置，或通过反复试验指定表面细节等级和阈值方法），检查生成的表面是否反映荧光信号。要指定兴趣区域，请选择通过裁剪X，Y，Z位置来呈现的区域和时间。 注意:选择 整个图像的过程最终将使整个电影的调整和阈值。 </li><li>再次单击向前箭头按钮生成曲面。然后通过更改滤波器中的直方图来调整阈值，以对应于膜信号。 </li><li>表面生成后，在"曲面/设置"中选择曲面，以便可视化表面信号，并通过推动双重打击来完成表面生成过程箭头按钮（ 图3 B ）。 </li><li>去Surface |编辑并单击蒙版 属性中的 全部 遮罩以生成蒙版通道图像。 </li><li>点击确定后，屏幕通道窗口将自动打开;选择GFP通道，将表面外部的体素设置为最大信号强度值（在编辑|显示调整中找到 ）和表面内的体素到最小GFP强度值（也见于 编辑|显示调整 ），在面罩设置中生成膜信号的掩码。 </li><li>由于膜信号的掩模将在体视图中显示，请使用图像处理|生成反转图像对比变化|反转 （ 图3 C ）。 </li></ol></li><li>从反面图像中检测斑点。 注意:此部分允许每个单元格的中心标志着一个独特的地点，跟踪了太空和时间。 <ol><li>选择超越场景|斑点|创建 ;在算法设置中，选择仅划分兴趣区域， 最终完成整个图像的处理和跟踪点（随着时间的推移） 。 </li><li>点击向前箭头并指定感兴趣的区域;选择区域和时间跟踪斑点;继续按向前箭头，选择屏蔽通道作为源通道，并确定点的估计XY直径。 </li><li>点击向前箭头按钮将自动打开一个过滤器窗口;通过改变直方图中的值来调整点检测阈值，以在每个单元格的中心产生单个点（ 图3 D ）。 </li><li>再次点击向前箭头按钮将打开算法窗口，选择自回归运动跟踪模型 20，并指定最大跟踪di立场和最大间隙大小，以连接在短时间内变得不连续的轨道。 </li><li>再次点击向前箭头按钮将打开"过滤器"窗口，通过调整直方图来设置跟踪持续时间阈值，以便跟踪每个单元格。单击双向向前箭头按钮将完成点检测。 </li><li>为了纠正组织漂移，请点击Spots |轨道编辑器，并选择正确的漂移 。 算法窗口将自动打开;选择平移漂移，包括整个结果和正确的对象位置 。 </li></ol></li><li> 生成有利位置进行定量分析。 注意:程序的Scatterplot是用于生成具有多个对象的多维（X，Y，Z，颜色和缩放）图形的工具。这些图可用于查看许多单元格移动随时间变化的趋势。 <ol><li>使用"2D"或"3D"绘制单元格跟踪数据Vantage-Add New Vantage绘图菜单（ 图3 E和3F ）。 </li><li>选择卷或点，然后选择创建/类别和绘图类型 </li><li>调整绘图值以生成不同的有利图。 </li><li>使用快照功能导出图形。 注意:统计数据也可以作为电子表格导出，以进一步进行定量分析，方法是点击绘图数字区域保存 。 </li></ol></li></ol>

<ol>
	<li>Ray, H. J., Niswander, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+tissue+fusion+during+development.">Mechanisms of tissue fusion during development.</a> Dev. Camb. Engl. 139, 1701-1711 (2012).</li><li>Dudas, M., Li, W. -. Y., Kim, J., Yang, A., Kaartinen, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palatal+fusion+-+where+do+the+midline+cells+go?+A+review+on+cleft+palate,+a+major+human+birth+defect.">Palatal fusion - where do the midline cells go? A review on cleft palate, a major human birth defect.</a> Acta Histochem. 109, 1-14 (2007).</li><li>Bush, J. O., Jiang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palatogenesis:+morphogenetic+and+molecular+mechanisms+of+secondary+palate+development.">Palatogenesis: morphogenetic and molecular mechanisms of secondary palate development.</a> Development. 139, 231-243 (2012).</li><li>Lan, Y., Xu, J., Jiang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+Molecular+Mechanisms+of+Palatogenesis.">Cellular and Molecular Mechanisms of Palatogenesis.</a> Curr Top Dev Biol. 115, 59-84 (2015).</li><li>Taya, Y., O'Kane, S., Ferguson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+cleft+palate+in+TGF-b3+knockout+mice.">Pathogenesis of cleft palate in TGF-b3 knockout mice.</a> Development. 126, 3869-3879 (1999).</li><li>Kim, S., 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=Convergence+and+Extrusion+Are+Required+for+Normal+Fusion+of+the+Mammalian+Secondary+Palate.">Convergence and Extrusion Are Required for Normal Fusion of the Mammalian Secondary Palate.</a> PLOS Biol. 13, 100122 (2015).</li><li>Yoshida, M., 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=Periderm+cells+covering+palatal+shelves+have+tight+junctions+and+their+desquamation+reduces+the+polarity+of+palatal+shelf+epithelial+cells+in+palatogenesis.">Periderm cells covering palatal shelves have tight junctions and their desquamation reduces the polarity of palatal shelf epithelial cells in palatogenesis.</a> Genes Cells. 17, 455-472 (2012).</li><li>Fitchett, J., Hay, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medial+edge+epithelium+transforms+to+mesenchyme+after+embryonic+palatal+shelves+fuse.">Medial edge epithelium transforms to mesenchyme after embryonic palatal shelves fuse.</a> Dev. Biol. 131, 455-474 (1989).</li><li>Akira, N., 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+expression+of+TGF-beta3+for+epithelial-mesenchyme+transdifferentiated+MEE+in+palatogenesis.">The expression of TGF-beta3 for epithelial-mesenchyme transdifferentiated MEE in palatogenesis.</a> J Mol Hist. 41, 343-355 (2010).</li><li>Sani, F. V., 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=Fate-mapping+of+the+epithelial+seam+during+palatal+fusion+rules+out+epithelial-mesenchymal+transformation.">Fate-mapping of the epithelial seam during palatal fusion rules out epithelial-mesenchymal transformation.</a> Dev. Biol. 285, 490-495 (2005).</li><li>Jin, J. -. Z., Ding, J. <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+cell+migration,+transdifferentiation+and+apoptosis+during+mouse+secondary+palate+fusion.">Analysis of cell migration, transdifferentiation and apoptosis during mouse secondary palate fusion.</a> Development. 133, 3341-3347 (2006).</li><li>Xu, X., 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=Cell+autonomous+requirement+for+Tgfbr2+in+the+disappearance+of+medial+edge+epithelium+during+palatal+fusion.">Cell autonomous requirement for Tgfbr2 in the disappearance of medial edge epithelium during palatal fusion.</a> Dev. Biol. 297, 238-248 (2006).</li><li>Carette, M. J., Ferguson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+medial+edge+epithelial+cells+during+palatal+fusion+in+vitro:+an+analysis+by+Dil+labelling+and+confocal+microscopy.">The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by Dil labelling and confocal microscopy.</a> Development. 114, 379-388 (1992).</li><li>Muzumdar, M., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li><li>Dassule, H., Lewis, P., Bei, M., Mass, R., McMahon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+regulates+growth+and+morphogenesis+of+the+tooth.">Sonic hedgehog regulates growth and morphogenesis of the tooth.</a> Development. 127, 4775-4785 (2000).</li><li>Eisenhoffer, G., 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=Crowding+induces+live+cell+extrusion+to+maintain+homeostatic+cell+numbers+in+epithelia.">Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia.</a> Nature. 484, 501-546 (2012).</li><li>Riedl, 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=Lifeact:+a+versatile+marker+to+visualize+F-actin.">Lifeact: a versatile marker to visualize F-actin.</a> Nat Methods. 5, 605-607 (2008).</li><li>Riedl, 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=Lifeact+mice+for+studying+F-actin+dynamics.">Lifeact mice for studying F-actin dynamics.</a> Nat Methods. 7, 168-169 (2010).</li><li>Can, 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=Attenuation+correction+in+confocal+laser+microscopes:+a+novel+two-view+approach.">Attenuation correction in confocal laser microscopes: a novel two-view approach.</a> J Microsc. 211, (2003).</li><li>Veenman, C., Reinders, M., Backer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+motion+correspondence+for+densely+moving+points.">Resolving motion correspondence for densely moving points.</a> IEEE Trans Pattern Anal Mach Intell. 23, 54-72 (2001).</li><li>Udan, R., Piazza, V., Hsu, C., Hadjantonakis, A., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+imaging+of+cell+dynamics+in+mouse+embryos+using+light-sheet+microscopy.">Quantitative imaging of cell dynamics in mouse embryos using light-sheet microscopy.</a> Development. 141, 4406-4414 (2014).</li><li>Stoller, 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=Cre+reporter+mouse+expressing+a+nuclear+localized+fusion+of+GFP+and+beta-galactosidase+reveals+new+derivatives+of+Pax3-expressing+precursors.">Cre reporter mouse expressing a nuclear localized fusion of GFP and beta-galactosidase reveals new derivatives of Pax3-expressing precursors.</a> Genesis. 46, 200-204 (2008).</li></ol>

作者没有什么可以披露的。

使用3D器官外植体培养的组织形态发生的活体成像可以提供关于固定组织切片的常规染色分析中不能显示的细胞过程的详细信息。使用小鼠胚胎中性腭裂的体外外植体培养，我们观察到几个有趣的细胞行为，导致我们提出了一种涉及上皮收敛和细胞挤出的腭裂融合的新机制。 在这种研究中的一个常见挑战是通过长时间的连续激光激发进行光漂白。在我们的研究中，使用...

组织融合是多器官发育的重要一步。主要的人类出生缺陷如唇腭裂，脊柱裂和心脏畸形可能会导致从在组织融合1缺陷。小鼠二级腭融合已被广泛研究，以确定细胞和分子机制发展2，3，4控制组织融合。在小鼠中，二尖瓣发育开始于E11.5附近，其中每个双侧上颌过程均产生二级腭骨架。腭架的初始生长沿着舌头垂直发生，直到大约E14.0，此时腭架在舌头上方水平上升。中度指导的生长导致两个腭搁架的上皮的物理接触，形成中线上皮（MES）在E14.5。必须从中间腭架之间移除插入的MES，以允许间充质融合，并通过E15.5 3开发完整的，完全融合的中腭。 如何在两个单独的腭架之间形成共享的上皮MES细胞层，然后移除以实现间充质融合，一直是腭发育的中心问题。基于小鼠组织学和电子显微镜（EM）研究，外植体培养研究和功能小鼠遗传学实验，几个基本细胞行为已经涉及到这个过程。从每个货架腭的内侧边缘上皮MEE丝状伪足状突起便于初始接触5， 图6，随后将这些上皮细胞的嵌入到共享单个MES 6,7。删除生成的共享MES已被提议通过三个非排他性机制进行。采用组织学观察和离体谱系用活体染料示踪的早期研究表明，MES可能通过上皮被除去以MES细胞的间充质转换（EMT）8,9，虽然最近，上皮细胞的遗传谱系追踪已升高的不确定性，以上皮细胞的长期贡献腭间质架10，11，12。凋亡细胞的数量显著，并在他们的人数在某些突变体不能进行适当的味觉融合的减少，导致了想法，凋亡可能是MES溶解2,3的主要驱动力。最后，最初基于涉及上皮标记和进行时间点静态观察的研究MES细胞提出在口鼻及前后尺寸11，13迁移，但这样的动态细胞行为最初未经证实的由于无法观察他们在活组织腭。最近，我们能够通过开发一种新的活体成像方法直接观察这些行为，该方法结合了荧光标记的小鼠遗传方法与外植腭架的共聚焦活检。 首先，腭融合过程中可视化腭上皮细胞动态细胞行为，我们通过杂交ROSA26-mTmG FLOX小鼠Keratin14-CRE小鼠14,15生成的上皮特异性报道小鼠。所产生的胚胎的腭外植体培养物的共聚焦活体成像证实了一些先前提出的细胞行为，并在融合过程中鉴定了新的事件6 </SUP&gt;。上皮细胞膜突起先于初始细胞 - 细胞接触，然后通过细胞插入和鼻孔细胞置换上皮收敛。值得注意的是，我们还发现，电池挤出，报道在上皮内环境稳定中发挥重要作用的过程，是一个重大的机构驱动鼠标继发腭融合6,16。该成像方法可与其他记者行一起使用;我们利用Lifeact-mRFPruby转基因小鼠17，18在融合过程来检查肌动蛋白细胞骨架的动态。其他记者也可以用来观察腭部融合的其他具体方面，这种方法可以适用于激光扫描共焦显微镜或旋转盘共焦显微镜，这取决于成像需要和显微镜可用性。现场影像越来越成为发育生物学中的基石方法。班驳轻微地，颅面形态发生是复杂的，影响面部的人类出生缺陷是常见的。这种共焦现场成像方法将有助于更好地了解潜在的基本发育机制以及人类颅面异常的起源。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12</td><td>Life technology</td><td>11330-032</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life technology</td><td>16000-044</td><td></td></tr><tr><td>L-glutamine</td><td>Life technology</td><td>25030-081</td><td></td></tr><tr><td>L-Ascorbic acid</td><td>Sigma</td><td>A4544-100G</td><td></td></tr><tr><td>Pennicillin/Streptomycin</td><td>Life technology</td><td>15140-122</td><td></td></tr><tr><td>Low melting agarose</td><td>BioExpress</td><td>E-3111-125</td><td></td></tr><tr><td>35 mm glass bottom dish</td><td>MatTek</td><td>P35G-1.5-10-C</td><td></td></tr><tr><td>Petrolieum Jelly (Vaseline)</td><td>Sigma</td><td>16415-1Kg</td><td></td></tr><tr><td>&lt;strong&gt;Mice&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Keratin14-cre&lt;/em&gt;</td><td></td><td>MGI: J:65294</td><td>Allele = Tg(KRT14-cre)1Amc</td></tr><tr><td>&lt;em&gt;ROSA26&lt;sup&gt;mTmG&lt;/sup&gt;&lt;/em&gt;</td><td></td><td>MGI: J:124702</td><td>Allele = Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo</td></tr><tr><td>&lt;em&gt;Lifeact-mRFPruby&lt;/em&gt;</td><td></td><td>MGI: J:164274</td><td>Allele = Tg(CAG-mRuby)#Rows</td></tr><tr><td>&lt;strong&gt;Microscope&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>White Light SP5 confocal microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Cell Observer spinning disk confocal microscope</td><td>Zeiss Microscopy</td><td></td><td></td></tr></tbody></table>

live imaging of mouse secondary palate fusion

二级腭架融合形成完整的二级腭是哺乳动物发育的关键过程，其破坏可导致腭裂性腭裂，这是人类常见的先天性异常现象。二次腭融合已被广泛研究，导致几个可能介导该过程的细胞机制。然而，这些研究主要在开发期间的进行时间点或在静态时间点分析的固定外植体培养物中对固定的胚胎组织进行。静态分析限于动态形态发生过程的分析，如腭裂融合，以及什么类型的动态细胞行为介导腭融合不完全了解。这里我们描述了一种用于小鼠胚胎体外二次腭融合的活体成像方案。为了检查腭裂融合的细胞行为，上皮特异性角蛋白14 -cre用于标记ROS中的腭上皮细胞A26-mTmG flox记录胚胎。为了可视化丝状肌动蛋白，使用Lifeact-mRFPruby报道小鼠。通过解剖最近粘附的胚胎期（E）14.5期胚胎的次级腭架并在玻璃底盘上的含琼脂糖的培养基中培养以使用倒置的共聚焦显微镜成像来进行中度腭融合的活体成像。使用这种方法，我们检测到在二次腭融合期间的各种新型细胞行为。对不同细胞行为在空间和时间上的协调的欣赏大大有助于我们对这种动态形态发生过程的理解。该方案可应用于突变小鼠系，或用药理学抑制剂处理的培养物，以进一步提高对二次腭融合如何控制的理解。

腭外植体活体成像显示多种细胞过程介导腭裂融合6 。两个上皮细胞之间的初始接触由膜突起（ 图 2B-2E ）制成。当两个上皮层相遇时，它们形成多细胞分层MES，然后插入以通过上皮收敛形成整合的单细胞层MES（ 图 2A-2E和图 2K-2O ）。较深Z水平的上皮...

Watch this Scientific Journal Video about Live Imaging of Mouse Secondary Palate Fusion at JoVE.com

Live Imaging of Mouse Secondary Palate Fusion

在这里，我们提出了使用共聚焦显微镜的小鼠二次腭融合的活体成像方案。该方案可以与各种荧光报告小鼠系列结合使用，并可与途径抑制剂一起用于机械性的洞察。该协议可适用于其他开发系统中的实时成像。

小鼠第二腭融合的实时成像

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

live-imaging-of-mouse-secondary-palate-fusion

Research

JoVE Journal

Developmental Biology

7.5K 次观看.  University of California San Francisco. 在这里，我们提出了使用共聚焦显微镜的小鼠二次腭融合的活体成像方案。该方案可以与各种荧光报告小鼠系列结合使用，并可与途径抑制剂一起用于机械性的洞察。该协议可适用于其他开发系统中的实时成像。 

视频: Live Imaging of Mouse Secondary Palate Fusion

Method of Studying Palatal Fusion using Static Organ Culture

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that adheres to form the midline epithelial seam (MES). The theories suggest that MES cells follow an epithelial to mesenchymal transition (EMT), apoptosis and migration, making a fused palate 1. Complete disintegration of the MES is the final essential phase of palatal confluence with surrounding mesenchymal cells. We provide a method for palate organ culture. The developed in vitro protocol allows the study of the biological and molecular processes during fusion. The applications of this technique are numerous, including evaluating responses to exogenous chemical agents, effects of regulatory and growth factors and specific proteins. Palatal organ culture has a number of advantages including manipulation at different stages of development that is not possible using in vivo studies.

Studies of palate development are motivated by the incidence of cleft palate, a birth defect that imposes a tremendous health burden and can leave lasting disfigurement. We demonstrate here a technique to culture palatal shelves that can be used to study different signaling pathways involved in palatal development and fusion.

使用静态器官培养研究腭融合的方法

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

科研

发育生物学

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

阿的Cre - LOX P为检测细胞融合重组方法在体内</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

生物工程

Longitudinal Intravital Imaging Through Clear Silicone Windows

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

通过透明硅胶窗进行纵向活体成像

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

癌症研究

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

神经科学

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

CCL5诱导小鼠骨膜骨骼干细胞迁移的实时成像

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

小鼠的引导肿瘤接种和多模式成像

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

作者聚焦:整合高分辨率活体成像和 MRI 以增强立体定向放射治疗计划

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

背侧皮褶窗室肿瘤小鼠模型在转化癌症研究中用于联合活体显微镜和磁共振成像

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

原代小鼠胚胎腭间充质细胞的分离和延时成像，以分析集体运动属性

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

通过双光子显微镜实时跟踪牙齿细胞

Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal stem cells and tooth regeneration. These progenitor populations actively divide, move, and differentiate to maintain tissue homeostasis and regenerate lost cells in a responsive manner. However, traditional analyses using fixed tissue sections could not capture the dynamic processes of cellular movements and interactions, limiting our ability to study their regulations. This paper describes a protocol to maintain whole mouse incisors in an explant culture system and live-track dental epithelial cells using multiphoton timelapse microscopy. This technique adds to our existing toolbox for dental research and allows investigators to acquire spatiotemporal information on cell behaviors and organizations in a living tissue. We anticipate that this methodology will help researchers further explore mechanisms that control the dynamic cellular processes taking place during both dental renewal and regeneration.

作者聚焦:了解成年小鼠牙齿组织更新和修复中的动态细胞行为 

Ex vivo live imaging is a powerful technique for studying the dynamic processes of cellular movements and interactions in living tissues. Here, we present a protocol that implements two-photon microscopy to live track dental epithelial cells in cultured whole adult mouse incisors.

使用 离体 实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal ...

小鼠第二腭融合的实时成像

摘要

探索更多视频

此视频中的章节

使用静态器官培养研究腭融合的方法

阿的Cre - LOX P为检测细胞融合重组方法在体内

通过透明硅胶窗进行纵向活体成像

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

CCL5诱导小鼠骨膜骨骼干细胞迁移的实时成像

背侧皮褶窗室肿瘤小鼠模型在转化癌症研究中用于联合活体显微镜和磁共振成像

背侧皮褶窗室肿瘤小鼠模型在转化癌症研究中用于联合活体显微镜和磁共振成像

背侧皮褶窗室肿瘤小鼠模型在转化癌症研究中用于联合活体显微镜和磁共振成像

原代小鼠胚胎腭间充质细胞的分离和延时成像，以分析集体运动属性

使用离体实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

小鼠第二腭融合的实时成像

摘要

探索更多视频

此视频中的章节

使用静态器官培养研究腭融合的方法

阿的Cre - LOX P为检测细胞融合重组方法在体内

通过透明硅胶窗进行纵向活体成像

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

CCL5诱导小鼠骨膜骨骼干细胞迁移的实时成像

背侧皮褶窗室肿瘤小鼠模型在转化癌症研究中用于联合活体显微镜和磁共振成像

背侧皮褶窗室肿瘤小鼠模型在转化癌症研究中用于联合活体显微镜和磁共振成像

背侧皮褶窗室肿瘤小鼠模型在转化癌症研究中用于联合活体显微镜和磁共振成像

原代小鼠胚胎腭间充质细胞的分离和延时成像，以分析集体运动属性

使用 离体 实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

使用离体实时成像研究小鼠牙齿更新过程中的细胞分裂和运动