Name: quantification of orofacial phenotypes in xenopus
Uploaded: 2014-11-06T15:00:00.000+00:00
Duration: 9 min 26 s
Description: Watch this Scientific Journal Video about Quantification of Orofacial Phenotypes in Xenopus at JoVE.com

启动资金来自VCU A·迪金森支持这项工作。 作者要感谢丹Nacu为他的艺术天分在创建示意图。

使用非洲爪蟾进行的所有实验已通过IACUC（协议＃AD20261）。 1.准备试剂及所需材料<ol><li>试剂: <ol><li>让1升的10倍MBS（修改巴特的盐）溶液22。添加氯化钠（880毫摩尔），氯化钾（10毫摩尔）， 硫酸镁 （10毫米），HEPES（50mM的，pH值7.8），和的NaHCO 3（25毫米）到1升的蒸馏水中。调节pH至7.8，用NaOH。 </li><li>使1升1倍的MBS稀释百毫升900毫升的蒸馏水中10倍的MBS溶液。添加0.7毫升的1M的CaCl 2溶液。 </li><li>使1升的0.1倍的MBS稀释100毫升于900ml的蒸馏水中1倍的MBS溶液。向1升的0.1倍的MBS溶液，在胚胎培养使用加入1毫升10毫克/毫升庆大霉素溶液。 </li><li>通过在100毫升10×的MBS溶解4毫升的5M的NaCl使高盐的MBS。然后加蒸馏水至总体积为1 L. </li><li>让70％的乙醇稀释100％Ethanol到70％乙醇中，用蒸馏水。 </li><li>使BMS-543通过制备10mM储备溶液在二甲基亚砜（DMSO）中。 </li><li>使4％多聚甲醛（PFA）通过加入4g多聚甲醛粉末至50ml蒸馏水。热的热板约65℃，此时加5 M氢氧化钠滴至溶液澄清。 <ol><li>一旦溶液清除，离火，加入50毫升2X PBS和100微升吐温20。允许冷却至室温冰上。调节pH值用HCl中7.2和7.5。 </li></ol></li><li>通过溶解8克氯化钠，0.2克的KCl，1.44克Na- 2 HPO 4，及0.24g的KH 2 PO 4在800毫升蒸馏水使用吐温（PBT）的磷酸盐缓冲盐溶液。使用盐酸，调节pH值至7.4。加2 ml吐温20。调整用蒸馏水终体积等于1升</li><li>使通过将1g的半胱氨酸在50毫升蒸馏水或0.1×MBS的半胱氨酸溶液。加入1.5毫升5 M氢氧化钠一次将pH调节至7.8。 </li><li>使10％的苯佐卡因原液加入10克苯佐卡因粉末至100毫升100％乙醇。对于成年男性和胚胎准备好固定的安乐死，稀释成0.05％的溶液。 </li><li>使睾丸存储溶液加入1 ml小牛血清的9毫升L15缓冲液中。 </li></ol></li><li>所需工具和设备: <ol><li>获得材料和设备的表中列出的设备， 如图1所示。 </li><li>修改吸管工具。放置一个玻璃巴斯德吸管的尖端插入从本生灯火焰。旋转枪头，直到其熔化，形成了一个圆形的密封端。 </li><li>扁平化模型粘土成的塑料培养皿的底部，平滑，以覆盖整个板（任何尺寸都可以使用）（ 图1Aiv）。 <ol><li>轻按直取笑针刺入粘土内衬盘以45°角。持针那里，缓慢移动的Petri菜水平，以创建一个郁闷线（ 图1Bi）。重复对行的期望数目。 </li><li>使用玻璃吸管工具末端，使粘土衬盘的每一行中的浅的圆形凹部放置胚胎头（ 图1Bii）和助剂在组织的胚胎而拍摄。 </li><li>对于摄影，补菜PBT（ 图1Biii）。使用之间，彻底清洗餐具和粘土表面用无菌水，接着用70％乙醇。 </li></ol></li></ol></li></ol> 2. 非洲爪蟾胚胎培养和抑制剂治疗<ol><li> 在非洲爪蟾卵体外受精培养: <ol><li>获得文化非洲爪蟾使用已发布的标准方法22,23胚胎。 </li><li>简要地说，在一个0.05％苯佐卡因溶液通过浸没安乐死成年男性。解剖睾丸并存储在睾丸的存储缓冲区。注成年女性，人绒毛膜促性腺激素（HCG），收集在高盐的MBS蛋和受精在体外用解剖睾丸（ 图2A，B）。 </li><li>在0.1倍的MBS在15℃下培养胚，直至所需阶段（根据非洲爪蟾的由新科普和法伯24,25的普通表期胚胎）到达。 <ol><li>在这里，培养的胚胎直到阶段22（24小时后的受精（高倍视野）），并在使用两对锋利的钳子的立体显微镜除去卵黄膜。移植胚与标准，一次性移液到含有30-40毫升0.1×MBS的干净的培养皿（直径为100mm）。 </li></ol></li></ol></li><li> 爪蟾胚胎的抑制剂处理（ 图2中示出）: <ol><li>填写一个24孔培养皿1毫升0.1倍MBS的使用校准移液管人必要的井。用细点大关划定井外器（ 图2D，插图）。使用该标记来也标注的内容被添加到每个孔中的24孔培养皿的盖子。在实验室的笔记本电脑将这些信息复制到一个24孔的设置页面。 </li><li>传送5-10胚胎成每使用一个标准的，一次性移液管（ 图2C）良好。放置在每个胚胎的数量相同井。 </li><li>补足或取出的0.1倍的MBS，以便它是与1ml的标记线（ 图2D）的水平。添加DMSO中的适当体积（以使DMSO的终体积为1％），并轻轻摇动。要小心，不要添加DMSO太近胚胎，并避免胚胎与缓冲的表面相接触。 </li><li>添加化学品的适当体积的指定孔中，轻轻地再摇动。这里，加入1微升的10mM的BMS-453（视黄酸受体拮抗剂）和图9微升DMSO中至1ml 0.1倍的MBS。 </li><li>如果化学品是光敏感的，松散包装的小路TURE盘中铝箔。培养的胚胎在15℃培养箱中，直到所希望的阶段。在本实施例中，治疗的胚胎为16-18小时。 </li></ol></li><li>洗出的胚胎治疗和饲养，直到蝌蚪阶段: <ol><li>除去二分之一至四分之三用一次性移液管将溶液。要小心，不要打扰胚胎。使用新的移液管，填充井与0.1X MBS。重复此3-6次。 </li><li>转移胚胎到装有100毫升的0.1倍的MBS用庆大霉素（1毫升/升）的大陪替氏培养皿（直径150mm）。孵化的胚胎在15℃，直到它们到达阶段42-45（82-100高倍视野）。 </li><li>每天都在变化的0.1倍MBS含庆大霉素溶液中，并去除死胚迅速，以防止污染其他胚胎或死亡。记录在实验室笔记本死胚的数量。 </li></ol></li><li>在多聚甲醛固定胚胎（PFA）: <ol><li>修正阶段42和45（82和100之间的胚胎高倍视野，分别）通过将它们转移到含有1-2毫升0.1×MBS的一个新的24孔培养皿中。标签盖用作为治疗建立上述相同的信息（第2.2.1节）。 </li><li>从每口井尽可能删除尽可能多的0.1倍MBS，同时还留下涵盖胚胎，并确保最小的接触，以防止受伤。加入1-2毫升0.05％苯唑卡因溶液，0.1倍MBS和孵育，直到胚胎不再响应的刺激。 </li><li>加1-2毫升4％PFA的。或者，将胚胎在标有微量离心管的固定，如果愿意的话。 </li><li>孵化的胚胎在PFA中24小时，在4℃下在旋转摇床上。通过在3-5小时内进行3-4次洗涤与PBT删除PFA。 </li></ol></li></ol> 3.拍摄非洲爪蟾蝌蚪的口面区<ol><li>预拍摄准备（这被示于图3）: <ol><li>放置在一个大的培养皿中固定的胚胎（150毫米直径）含有约100ml的PBT。 </li><li>使两个切口从身体（ 图3A的实黑线）切断的头部。首先，将与一对钳子的胚胎，并在肠道内的后部侧的切口，用无菌解剖刀（ 图3B）。做对肠道，心脏附近的前侧的第二切口，彻底清除头部（ 图3C）。 </li><li>移液管被切断胚头到使用标准的，一次性移液管粘土内衬盘（1.2.2部分）。 <ol><li>对于正面的看法，使用两对钳胚胎头后侧向下凹陷的圆形内侧的位置。使用镊子操纵两个胚胎头部及周边粘土，使得它们面对摄像机和不向后或向两侧（ 图3D）倾斜位置的胚胎。轻推头部周围使用镊子和/或玻璃周围的泥土吸管工具以固定头就位。 </li><li>横向视图，使用镊子来定位的圆形凹内胚头，这样他们就在自己身边，对着同一个方向，并且是平贴在粘土。操纵胚胎头部及周边粘土，使所有的胚胎的水泥腺体都以相同的角度（ 图3E）向下放置。使用绘制的戏弄针为指导，以确保准确性取侧卧测量时的行。 </li></ol></li></ol></li><li>捕捉蝌蚪头部的图像: <ol><li>拍摄使用任何解剖显微镜与一个附加的数码相机和相应的摄像头软件，蝌蚪的头。使用也可以保持恒定的所有胚胎的最高放大倍数。 </li><li>居中的胚胎和面对它们都在相同的方向。采集使用最佳的照明条件，使该口面部区域的最准确的图像的图像。位置灯，以避免过多的阴影。图像保存为.TIFF与尽可能高的分辨率的文件。 </li></ol></li></ol> 4.测量和分析面部大小尺寸在非洲爪蟾蝌蚪<ol><li>测量口面部尺寸（总结于图4）: <ol><li>确定脸部宽度。确定在每个眼睛的腹侧部分符合面部（ 图4A中，箭头）的周围的各点，并测量这些点（ 图4A中 ，红色线）之间的距离。 </li><li>确定的面部的高度。首先，通过测量每一只眼睛之间的距离，并计算半路标记确定的中线。接下来，绘制标注眼睛的背最边缘和最背点的水泥腺（ 图4B，白线）的水平线。在中线，测量水平线（ 图4B中 ，红色线）之间的距离。 </li><li>决定T他颜面部地区。使用相同的水平线标记眼睛和水泥腺引导面积测量的背缘（ 图4C中 ，白线）。开始在水平标线水泥压盖的上满足面（ 图4CA）的左侧的周围的点。 <ol><li>沿面包围的眼，直到其中的水平线标记眼睛的顶端满足面（ 图4CB）的周围的点的边缘跟踪。继续沿此背最水平线跟踪，直到它遇见右侧面（ 图4CC）的周边的点。 </li><li>迹向下沿面包围的眼，直到其中的腹侧最水平线标记水泥腺的顶部满足右侧面（ 图4CD）的周边的点的边缘。继续沿着这条底部水平线UNT追踪金正日满足的地方开始跟踪点。使用照片编辑软件来计算跟踪内的区域。 </li></ol></li><li>确定吻长。上侧的图像，使该标记的眼睛的前一个垂直线。接着，测量从前面的最点所在的面满足水泥腺垂直线标记的眼（ 图4D）的前部的背侧边缘的水平距离。 </li><li>确定嘴巴的宽度。测量左和右的点之间的水平距离，其中所述张口相遇（ 图4E）的顶部和底部的"嘴"。这可以被认为是青蛙相当于唇合缝的。 </li><li>确定嘴圆。计算圆口作为一个逆纵横比，其中（4 &#8197; × &#8197; [区域]）/（π &#8197; × &#8197; [长轴] 2 </sup&gt;）。 <ol><li>用套索工具在照片编辑器来跟踪嘴开口的边缘， 如图4F。选择主工具栏中的分析，并选择测量。可替换地，从区域和嘴开口的直径的测量计算圆度。 </li></ol></li></ol></li><li>面部尺寸的数据分析: <ol><li>输入的面部尺寸的测量值成一个数据分析程序，以治疗组与对照进行比较。确定每个测量两个实验组和对照组，以创建柱状图的平均值。确定以创建误差棒的标准偏差。进行Student的t检验进行统计比较组。 注:此数据可以说是相当的变量，由于胚胎中略有不同速率的发展。 </li></ol></li></ol>口面形状和形态测量5.定量分析<ol><li>广场的地标和CREA德的地标坐标文件（ 图5A所示）。 <ol><li>选择的地标，使得它们捕获目标图像的形状，并可以反复放置在相同的相对位置在所有样品中被分析。如果结构没有在所有样品中存在，不会对这种结构的一个里程碑。 <ol><li>这里，将24地标来表示与识别标志，如眼睛，鼻孔和嘴的面中部。为了保持一致性，地方以前放置​​的标志（例如，口标志性建筑，图5AI）之间的中间标志性建筑。 </li></ol></li><li>捕捉具有里程碑意义的坐标，打造"具有里程碑意义的坐标文件"。打开一个蝌蚪面（例如，第一控制胚胎）的所述第一图像。 <ol><li>选择主工具栏上的插件选项卡，然后从下拉菜单中选择Pointpicker。选择所需的位置加分标签和地点地标建筑地标将显示为彩色的十字架（ <strong&gt;图5AI）。 </li><li>通过选择移动工具，十字移动位置后的标志性地点。当所有地标都被放置在图像上，选择主工具栏上的显示结果选项卡，然后选择Show（ 图5Aii），以显示具有里程碑意义的坐标，并复制到电子表格程序（ 图5Aiii）。 </li><li>当粘贴从这个第一胎到电子表格程序，这些坐标，离开上述数据的空行。在这个空行（第1行），键入"LM ="与样品中的标志性建筑多项B列。在这里，输入"LM = 24"，因为24标志被创建（ 图5Aiii，红色框）。 </li><li>在下面的数据点，类型的行的"ID ="同一个名称识别所述样品。在这里，在图5Aiii，输入"ID = CON1"，因为这是具有里程碑意义的坐标，第一控制胚胎（ 图5Aiii，红色箭头）的数据。 </li><l我&gt;重复此过程，在实验中（实验和控制）每个胚胎的脸，直到所有的标志性坐标被输入到电子表格中。 注:重要的是，每个胚胎中给出的"ID ="行的唯一名称。这里，例如，下一组的地标坐标给出"CON2"的ID，因为它是在对照组中所述第二胎。 </li><li>整个第二列和第三列复制到一个文本文档，并保存为.txt文件。 </li></ol></li></ol></li><li>设置用于数据分析的形态的软件程序（在图5B中示出）。 <ol><li>在这里，选择在形态软件程序的主菜单上创建新的数据集。将该文件命名，并在相应的框中设置的数据（例如，"维甲酸抑制"和"面部标志性建筑"，分别），在弹出的界面（ 图5Bi）。导入文件作为TPS，和SELECT坐标以创建数据集（ 图5Bi，红色箭头）的文本文件。 </li><li>数据对齐和普鲁克适合: <ol><li>执行普鲁克配合和调整。突出显示在工程树选项卡中设置的数据，主工具栏上选择预赛和新飞度的Procrustes在下拉菜单中。 </li><li>选择对齐由主轴，并选择执行普鲁克适合在对话框（ 图5Bii，红色箭头）的底部。返回预赛下拉菜单中，选择生成协方差矩阵（ 图5Biii），然后选择执行。 </li><li>查看该协方差矩阵的相关性，在结果选项卡。 </li></ol></li><li>创建一个"分类文件"通过分配中设置为相应的分类变量的数据中的每个样品，以区分它是否属于在实验组或对照组。 <ol><li>标签在电子表格中两列。标记第一柱上进行的标题n，其中"ID"和输入相同的ID的每个样品给出（例如，CON1，RARINHIB1，等等; 图5Biv）。标记为"治疗"和输入的第二列的标题为每个小区对应于在相邻的ID细胞中列出的样品的治疗组。 </li><li>在这里，用"控制和RAR抑制剂"标签的分类（图5Biv）。复制到一个文本文件并将其保存为.txt文件。进口分类变量进入形态的软件程序。选择匹配标识符，并打开文本文件（ 图5BV）。 </li></ol></li></ol></li><li>创建一个主成分分析（PCA）的散点图通过选择变异下拉菜单中，选择主成分分析（ 图6AI）。接下来，选择图形选项卡可以看到另外三个选项卡:PC形态的变化，特征值，和PC的分数（ 图6Aii）。电脑评分卡显示Bivariate的普鲁克适合具有里程碑意义的数据（ 图6Aii）的主成分散点图。 <ol><li>要更改主成分绘制，带来了在剧情空间的弹出菜单，然后选择所需的轴来改变（ 图6Aiii，红色箭头）。 </li><li>选择颜色数据点的工具（ 图6Aiii）。选择分类以前进口的（ 第5.2.2节 ）中出现，以确定每个类别（ 图6Aiv）的颜色在第二个弹出式菜单。 </li><li>查看方差在结果选项卡（ 图6AV）各主成分的捕获量。导出图像为.bmp文件。 </li></ol></li><li>通过比较选项卡（ 图6Bi）下选择判别分析建立在形态学软件程序判别函数分析（DFA）改造电网。在弹出的菜单中，确保适当的数据集是SELected和数据的类型是普鲁克配合坐标。 <ol><li>突出显示要比较组的配对，并运行1000排列测试（ 图6Bii）。 </li><li>在图形，查看形状差异选项卡（ 图6Biii）坐标的矢量地图。如果图像被反转时，调出弹出菜单中的情节空间翻转图中的正确方向（ 图6Biii）。 </li><li>矢量的方向反射从所述第一组中的形状差异相比于第二，它是在运行分析（ 图6Bii）之前显示在判别功能菜单。到反向矢量的方向，调出在剧情空间的弹出菜单和改变比例因子的符号，以便它是负的（ 图6Biii，ⅳ）。比例值的值被设置为10的一个因数最能代表betwee界标位置的统计差异n中的两组不变形，并且通常保持默认（ 图6Biv）。 </li><li>另外，在弹出菜单，图形改变为变换网格叠加在载体上的网格（ 图6Biii，v）中 。 </li><li>指定的格子中的弹出式菜单中的水平和垂直线的数目（ 图6Biii，v）中 。 </li><li>图表导出为.bmp文件。 </li><li>查看结果选项卡（ 图6Bvi）下的Procrustes和Mahalinobis距离和p值。 </li></ol></li><li>附加的形态分析，这对于一个以上的治疗组的评估非常有用<ol><li>创建主成分分析变换网格。在PC形态的变化选项卡显示的形状变化占方差每个样本组中的矢量地图。使用弹出菜单中的绘图区，以正确的定位和形象载体叠加到电网改造。设置比例因子上对应于对照组的估计中心的PC分值曲线图的x轴的值。图像导出为.bmp文件。 </li><li>创建一个CVA散点图。在主工具栏上的比较选项卡中，选择规范变量分析，突出分类变量集以前进口的（参见第5.2.3节 ）。选择1000迭代置换试验，并选择执行。选择CV得分选项卡以查看二元CVA散点图。此基础上，上载的分类器的颜色编码。改变通过打开弹出式菜单中的情节空间的配色方案（参见5.3.3节 ）。导出为.bmp文件。 </li><li>创建一个CVA改造电网。比较两组以上时，可生成的CVA结果的变换格。在图形选项卡上，选择CV造型变化可视化的具有里程碑意义的坐标变换网格。调出弹出菜单中的情节空间改变该矢量的方向上，叠加的结果到一个转换的网格，并指定网格线的数量。图表导出为.bmp文件。 </li></ol></li></ol>

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作者什么都没有透露。

非洲爪蟾已成为解剖的发展机制基本颜面部开发的有用工具;然而，目前没有协议描述该区域的青蛙大小和形状的变化。此处描述的方法将通过允许在非洲爪蟾和其他脊椎动物口面部表型的更严格的量化显著向口面发展领域。 正确地执行该协议的第一，最关键的方面是既准确和可重复地测量人脸的尺寸和确​​定界标放置的能力。为此，这是至关重要的胚胎面以...

其中最普遍的和破坏性的类型的人的出生缺陷是指那些影响嘴和面部，如口面裂1。与颜面部畸形患儿的结构进行多次手术整个生命周期，并与面部抽搐，言语，听力和吃饭问题挣扎。因此，促进新的研究中cranio-和口颌面发育是至关重要的，以防止这些类型的出生缺陷和治疗的人类。 非洲爪蟾已作为解剖理事颅面发展的机制的新工具（一些实例包括2,3,4- -11）。因此，定量的方法来分析发展的头这一物种的面部在大小和形状的变化可能是非常强大的3。 在这里，我们提出这样的方法;结合改编自非洲爪蟾的研究12的几何形态测量传统的三围尺寸</SUP&gt;和丰富的研究分析人类的面部形态13-15。此协议的目标是允许研究人员进行量化的面部大小和形状正常和异常发育过程中不同的表型口面之间进行区分。这个分析将允许细微颅面畸形如从基因和/或环境因素的协同作用所产生的那些之间的更好区分。此外，这种定量​​方法也可以揭示甚至略有改善的颜面部缺损或救援。因此，这使得它在分析潜在的治疗提供有益的指导。 我们在座的面部测量和几何形态测量的结合使得两者的大小及颜面部区域的形状比目前的方案有更全面的统计分析，这在很大程度上只使用一个或另一个15-18。此外，我们提出了一种简单的方式来评估两者的内侧和外侧的平面面对而不需要在当前的研究中13,19中使用先进的三维成像的设备。 我们证明该协议对非洲爪蟾用视黄酸受体抑制剂诱导颜面部畸形发展，中位数腭裂2,3处理的胚胎。口面区域，在这些胚的尺寸和形状的定量揭示变化的面中部是类似于人类具有类似腭裂和小鼠模型20,21。然而，该协议可以用于评估的其它化合物上颌面发育如天然物质，除草剂，或蛋白质，如生长因子的效应。另外，从通过丢失或功能实验增益基因表达的扰动引起的口面的尺寸和形状变化（使用反义吗啉代或保鲜盒/塔伦斯）也可以使用该协议进行定量。最后，我们开发了这个方法的specificaLLY评估爪蟾形态;然而，可以很容易地修改为任何脊椎动物的分析。其他应用还可能包括使用该协议的进化和生态学研究比较密切相关的物种。虽然我们在这里提供的例子中使用这个协议描述颜面部区域的分析，它可以很容易地进行修改，对其他地区，器官或结构的分析。 这口面量化协议将成为研究界的宝贵资源，以及一个优秀的教学工具，用于本科生的视频演示。

<table><tbody><tr><td>Dissecting microscope</td><td>Zeiss</td><td></td><td>fitted with AxioCamICC1 camera</td></tr><tr><td>Dumont #5 Inox forceps</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>Sterile, disposable scalpel</td><td>Sklar</td><td>06-2015</td><td></td></tr><tr><td>24-well plate</td><td>Fisher Scientific</td><td>087721</td><td></td></tr><tr><td>Standard Disposable transfer pipettes</td><td>Fisher Scientific</td><td>13-711-7M</td><td></td></tr><tr><td>150 mm x 15 mm Petri dishes</td><td>Falcon</td><td>351058</td><td></td></tr><tr><td>Incubators</td><td>Ectotherm</td><td></td><td>set to 15 &amp;deg;C or 20 &amp;deg;C</td></tr><tr><td>Modeling Clay</td><td>Premo, or other non-toxic modeling clay</td><td></td><td>in black or white</td></tr><tr><td>Straight teasing needle</td><td>Thermo Scientific</td><td>19010</td><td></td></tr><tr><td>Capillary Tubing (for needles)</td><td>FHC</td><td>30-30-1</td><td>Borosil 1.0 mm OD x 0.5 mm ID/Fiber, 100 mm each</td></tr><tr><td>Needle Puller, Model P-97</td><td>Sutter Instrument Co.</td><td>Needle Puller: P-97 Flaming/ Bown micropipette puller Filament: FB300B</td><td>For filaments, use Sutter 3.00 mm square box filaments, 3.0 mm wide.</td></tr><tr><td>Pipettemen</td><td>Gilson</td><td>F144802, F123600, F123602</td><td></td></tr><tr><td>BMS-453</td><td>Tocris</td><td>3409</td><td></td></tr><tr><td>DMSO</td><td>American Bioanalytical</td><td>AB00435-01000</td><td></td></tr><tr><td>Cysteine</td><td>Sigma-Aldrich</td><td>52-90-4</td><td></td></tr><tr><td>Paraformaldehyde powder</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Petri dishes</td><td>Falcom</td><td>353003, 351058</td><td>100 mm diameter and 150 mm in diameter</td></tr><tr><td>100% Ethanol</td><td>VWR</td><td>89125-170</td><td></td></tr></tbody></table>

quantification of orofacial phenotypes in xenopus

爪蟾已成为解剖理事颅面发展和缺陷的机制的一个重要工具。 A到量化口面的开发方法将允许颜面部表型废除后，更严谨的分析与物质，可以遗传或分子操纵基因表达或蛋白质的功能。使用胚胎头的二维图像，传统的大小尺寸，如口面的宽度，高度和区内─被测量。此外，胚胎口开口的真圆度测量，用于描述所述口的形状。也执行这些二维图像的几何形态测量，以提供变化的口面部区域的形状的一个更复杂的图。标被分配给特定点的口面部区域，并创建坐标。一个主成分分析方法，以减少具有里程碑意义的坐标主要成分是那么的歧视待遇组。这些结果显示为散点图，其中以类似的口面形状的个体聚集在一起。它也执行判别函数分析，该统计比较两个治疗组之间的地标的位置是有用的。该分析显示在变换格，其中变化的地标位置被看作向量。网格叠加在这些载体中，使得整经图案被显示，以显示其中显著地标位置发生了变化。在判别函数分析的形状的变化是基于一个统计测量值，并因此可以通过一个p值来评价。这种分析既简单又方便，只需要一个立体镜和免费软件，因此将是一个有价值的研究和教学资源。

此处，口面部的大小和形状的定量分析证明，比较与视黄酸受体抑制剂（RAR抑制剂），以未处理的对照处理的胚胎。胚胎均采用这种化学抑制剂的1微米的浓度从24级到30（26-35 HPF），冲了出去，并固定在42级（82 HPF）。然后将它们加工成在协议中所述进行分析。结果是原始数据，但在以前的出版物2,3观察结果一致。控制胚胎与车辆，DMSO处理的，和正常发育（ 图7AI，ⅱ）。与RAR...

Watch this Scientific Journal Video about Quantification of Orofacial Phenotypes in Xenopus at JoVE.com

Quantification of Orofacial Phenotypes in Xenopus

甲量化非洲爪蟾胚胎的口面的大小和形状的方法已被开发出来。在这个协议中，传统大小的测量相结合，与几何形态测量，以允许口颌面发育和缺陷的更复杂的分析。

口面表型的定量<em&gt;爪蟾</em

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

quantification-of-orofacial-phenotypes-in-xenopus

Research

JoVE Journal

Biology

Quantification of Orofacial Phenotypes in Xenopus

9.7K 次观看.  Virginia Commonwealth University. 甲量化非洲爪蟾胚胎的口面的大小和形状的方法已被开发出来。在这个协议中，传统大小的测量相结合，与几何形态测量，以允许口颌面发育和缺陷的更复杂的分析。 

视频: Quantification of Orofacial Phenotypes in Xenopus

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

了解早期器官使用简体<em&gt;原位</em&gt;杂交协议<em&gt;爪</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

科研

发育生物学

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

胚胎血管生成的可视化和定量分析<em&gt;热带非洲爪蟾</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

生物学

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

评估主要神经再生<em&gt;爪</em&gt;胚胎使用免疫染色

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

神经科学

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

颅面间质细胞的电

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

活的嗅觉途径的功能评价

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

颅面部发育中的SOX10可视化：使用枫转基因斑马鱼线时间推移共聚焦显微镜

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

在面部移植<em&gt;非洲爪蟾</em&gt;胚胎

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes. 
Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.

Regulators of melanocyte functions govern visible differences in the pigmentation outcome. Deciphering the molecular function of the candidate pigmentation gene poses a challenge. Herein, we demonstrate the use of a zebrafish model system to identify candidates and classify them into regulators of melanin content and melanocyte number.

使用斑马鱼鉴定色素沉着调节因子的反向遗传方法

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from ...

口面表型的定量

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了解早期器官使用简体

胚胎血管生成的可视化和定量分析

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾

评估主要神经再生

颅面间质细胞的电

活的嗅觉途径的功能评价

颅面部发育中的SOX10可视化：使用枫转基因斑马鱼线时间推移共聚焦显微镜

在面部移植

使用斑马鱼鉴定色素沉着调节因子的反向遗传方法