作者感谢亚历克斯Schier的亲切共享Zebrabow-M转基因株系，杰弗里·伯恩斯的pDEST载体和Renee西尔 - Daigle提供无微不至的关怀鱼设施和线路。 资金来源: 我们感谢来自NIDCR RO3DE024490和施赖纳斯医院慷慨的资金支持儿童（ECL）和博士后培训奖学金从圣地兄弟会儿童医院（LR和YK）。

马萨诸塞州总医院实验动物管理和使用委员会（IACUC）批准根据协议号＃2010N000106的所有程序。这是符合协会的评估和实验动物国际（AAALAC）的指导方针的认可。 1.试剂和材料准备<ol><li>制备1升的50X E3胚胎培养基（ 见表 1）和制备1升1X E3胚胎培养基（ 见表 2）。 </li><li>加入PTU 30毫克到1升1X E3的准备与N-苯基硫氧嘧啶（PTU）E3胚胎中期。搅拌O / N，以确保机动部队已完全溶解。存储在RT。 </li><li>为了制备链霉蛋白酶溶液，稀释150微升链霉蛋白酶（0.5毫克/毫升）的15毫升1X的E3。这一数额是足够的1培养皿。立即使用。 </li><li>准备甲基纤维素溶液。 <ol><li>制备E3-三卡因溶液通过混合75 ml的0.2％三卡因的用25ml的1X E3。煮沸3克甲基纤维素在75毫升E3-三卡因溶液。 </li><li>补足终体积到100毫升，E3-三卡因溶液。最终的甲基纤维素溶液是粘稠和不透明带黄色调。贮存:RT保护的光。 </li></ol></li><li>他莫昔芬的股票和感应解决方案<ol><li>通过将4-羟基在100％乙醇制备5mM的他莫昔芬原液。商店的股票在-80℃。 </li><li>准备新鲜的稀释他莫昔芬在1X E3的愿望，得到浓缩感应解决方案。注意！使用前请咨询MSDS。 </li></ol></li><li>制备1升0.2％三卡因的按表3中。 </li><li>获取共聚焦幻灯片。胶4玻璃罩（18×18）在一起，形成一个块，然后把2个街区，在24×60的玻璃罩相距2厘米，以便安装在中间的胚胎。 </li></ol> 2.胚胎制备<ol><li>转基因株系。 <br/&gt;注:Zebrabow-M是由亚历克斯Schier实验室的一份厚礼。 </li><li> SOX10:ERT2 Cre重组 注:使用标准的分子克隆方法ERT2 Cre重组和商业上获得重组克隆技术:生成靶向载体pDestTol2- SOX10。 <ol><li>结合5&#39;进入克隆（pENTR5&#39;- sox10p）含有7.2Kb的SOX10的启动子，一个网关中间克隆（PME-ERT2-Cre的），和一个3&#39;进入克隆（pENTR3&#39;-polyA的），其与透镜目的地LR反应矢量pDestTol2-α晶状体 :YFP。 </li><li>净化构建pDestTol2- SOX10:ERT2 Cre重组。共注入纯化构建体（100毫微克/微升）用TOL2转座的mRNA（50纳克/微升）到WT胚胎（Fo）的一个小区的阶段。 </li><li>屏幕F0成人创始人为种系传递的基础上强劲的黄色荧光F1胚胎。 </li></ol></li><li> SOX10:ERT2 Cre重组; Zebrabow-M <ol><li>异交的Zebrabow-M行（2.1.1）与SOX10:ERT2-Cre转基因。 </li><li>选择胚胎表现出较强的无处不在RFP的表达和YFP镜头标记和成长，直到他们成年。 </li></ol></li></ol> 3.胚胎收集<ol><li>建立了几个对SOX10的:ERT2 Cre重组; Zebrabow-M转基因斑马鱼下午5点和下午6点第1天之间。 </li><li>第二天，拉早上分频器和收集中午前后鸡蛋。 </li><li>他们转移到培养皿中，并新增15毫升链霉蛋白酶的解决方案，以dechorionate胚胎。 </li><li>孵育胚胎进行1〜5分钟，直到胚胎的约60％已被dechorionated。停止反应通过滗析链霉蛋白酶溶液，并用新鲜的E3介质洗胚胎3-4倍。 </li><li>孵育在E3的dechorionated胚胎在室温O / N，让他们发展，达到10体节阶段。每孵育50-60离合器胚胎，以避免不同的发育生长。 </li></OL&gt; 4.治疗<ol><li>检查光场显微镜下胚胎的发育阶段，以确保他们在10体节阶段。 </li><li>使用荧光显微镜与红色荧光蛋白（RFP）过滤，分离出亮红色的胚胎。 </li><li>加入10的羟基他溶液到培养皿μM和孵育所述分离的胚胎在28.5℃下进行3小时进行到Cre的重组的诱导。 </li><li>滗他莫昔芬溶液和E3洗胚胎几次。 注意！丢弃在一个特殊的生物废料容器中的他莫昔芬的解决方案。 </li><li>孵育胚胎在28.5°CO / N。 </li><li>当胚胎是大约28-29体节阶段（约24小时受精后），孵化的胚胎在28.5℃，在E3-PTU解决方案以后。 注:PTU是这里用来抑制在发育中的胚胎形成黑素细胞的。这种治疗是必要的，以避免荧光的由自体荧光黑色素细胞的信号失真。 </li><li>孵育胚胎在28.5℃， </li></ol> 5.胚胎选择<ol><li>以可视化的全面发展颅面结构中，选择红色信号和表达Cre的标记阳性（黄色视网膜）的强度的胚胎在4天后施肥。 </li><li>麻醉与E3 / 0.015％三卡因溶液的胚胎。当没有活跃的走势或鳍活动时，轻轻地捅了捅胚胎观察麻醉被确认。 </li></ol> 6.安装在甲基纤维素的胚胎<ol><li>传输用于成像所选胚胎成含有一滴甲基纤维素的培养皿中，并轻轻薰胚胎与甲基纤维素。 </li><li>在干净的共焦幻灯片，将一滴新鲜的甲基纤维素和使用微加载提示下拉内安装的胚胎。位置胚背采取谨慎不要碰直接胚胎。 </li><li>覆盖安装胚用25×25盖玻片并在必要时通过操纵盖玻片调节胚胎的位置。 </li><li>胚胎是现在已经准备好形象。 </li></ol>通过荧光显微镜7.分析<ol><li>使用20X干透镜物镜（数值孔径0.75;针孔大小2.0微米）对焦胚胎。 </li><li>按下显微镜L100按钮，并在软件中选择的通道。 </li><li>选择激光共聚焦设置按钮，然后单击"自动"，并关闭窗口前选择以下途径:CH1:CFP，CH2:GFP，CH3:RFP和mCherry </li><li>点击"删除互锁"按钮前，打开CH2和CH3。点击"播放"开始扫描。 </li><li>专注于感兴趣的结构，这是最好的开始半帧/秒的高电压（HV）150，然后相应地调整设置完成，直到所需的图像质量是交流hieved。招标书会比其他颜色更亮，从而调节高压。改变Z位置找到这两个YFP和RFP阳性细胞。 </li><li>一旦设定的设置，捕获胚胎的形象。保存你的照片进行图像处理的每个分割通道的共焦成像软件格式和TIFF格式。 </li><li>接下来，CFP成像，关闭CH2和CH3并打开通道1。重新调整设置如在步骤5，而不改变焦点区域。 注:CFP可真难以检测由于在蓝色通道的背景信号，可以通过调节针孔大小被规避。较大的针孔大小允许更好的检测荧光的减少信号/背景噪声比。然而，一个较大的针孔会降低共焦效果，影响图像质量。因此，仔细调整的设置是必需的以获得所需的图像质量和强度。 </li><li>捕获图像，并将其保存在两种格式（共聚焦软件和TIFF）进行图像处理。图像然后可以使用通用图像处理软件来合并层和所描述的水平和同事8提高颜色设置处理。 </li></ol>

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The authors declare that they have no competing financial interests.

阿尔新蓝和photoconvertible转基因株系上述相得益彰确定软骨和骨骼发育的复杂的过程。然而，器官在现场细胞迁移和组织长期以来一直推测，间接证明了，但从来没有显现。 Zebrabow-M的转基因系加上软骨特异性表达Cre允许同时实时观测参与骨和软骨形成所有这些不同的事件。这种技术允许的下颚生长缺陷在诸如罗宾序列，其中小颌是所描述的突出特征之一的条件的细胞和器官水平的研究。失败的增?...

颅面发展需要复杂的分子，细胞和组织的相互作用来驱动细胞的增殖，迁移和分化1,2,3。此严格调节和复杂的过程，受遗传和环境扰动，使得颅面畸形是其中最常见的出生畸形1-9。虽然外科手术仍然是主要的治疗方法为颅面畸形，了解发展的基础是至关重要的创新，未来的治疗方法。因此，研究的形态发生和机制中的收敛和延伸和细胞融合提供了新的见解的颅面骨1的形成。 颅神经嵴迁移和填充第一咽弓，进而形成配对下颌流程延伸到形成麦克尔软骨，这预示着下颌骨。形态Øf显示麦克尔软骨需要通过定向增殖，细胞分化和分化1,10软骨组织。然而，软骨细胞组织中的梅克尔软骨的生长和扩展的复杂仍不清楚。理解动态细胞行为是对理解影响下颌大小，如发育不全颌骨表型11先天性畸形的关键。 斑马鱼胚胎提供了对麦克尔软骨形态详细研究的许多发育和遗传的优势。其遗传易处理性，透明度， 离体快速发展的强大优势，通过实时成像6放贷很好的观察细胞的运动和组织。采用谱系追踪工具，如SOX10:枫转基因线，我们和其他人已经圈定了胚胎颅面骨1,5的神经嵴起源。 USI纳克的SOX10:ERT2 Cre重组与UBI:Zebrabow-M的转基因系，现在有可能颅面发育过程中，探索细胞运动的细节。所述Zebrabow-M，是转基因线工程与泛素启动子驱动的荧光团不同的表达，每个由lox位点8侧翼。该Zebrabow-M默认荧光红，表达RFP。诱导Cre的表达后，Zebrabow-M的构建重组和细胞表达不同的荧光团（RFP，CFP和YFP）创建在胚胎多光谱表达的组合。所有的子细胞从重组事件后的标记的细胞分裂，然后克隆标记，从而使细胞群，从不同的并列祖派生是克隆标记。由该克隆细胞标记，细胞增殖和迁移与克隆分辨率可以跟随（图1 和2）。

<table><tbody><tr><td>Pronase</td><td>Roche Life Sciences</td><td>10165921001</td><td>Prepare 500 &amp;mu;l stock aliquots at 50 mg/ml</td></tr><tr><td>Methylcellulose</td><td>Sigma-Aldrich</td><td>M0262</td><td></td></tr><tr><td>PTU (N-Phenylthiourea)</td><td>Sigma-Aldrich</td><td>P7619</td><td></td></tr><tr><td>Tricaine</td><td>Sigma-Aldrich</td><td>E10521</td><td></td></tr><tr><td>4-HydoxyTamoxifen</td><td>Sigma-Aldrich</td><td>T176</td><td></td></tr><tr><td>24 x 60 coverslips</td><td>Fisher Scientific</td><td>12-548-5P</td><td></td></tr><tr><td>18 x 18 coverslips</td><td>Fisher Scientific</td><td>12-540A</td><td></td></tr><tr><td>25 x 25 coverslips</td><td>Fisher Scientific</td><td>12-540C</td><td></td></tr><tr><td>pENTR5&#039;-TOPO TA Cloning Kit</td><td>Life technologies&amp;nbsp;</td><td>K591-20</td><td></td></tr><tr><td>pENTR/D-TOPO Cloning Kit&amp;nbsp;</td><td>Life Technologies</td><td>K2400-20</td><td></td></tr><tr><td>pENTR3&#039;-pA</td><td>Tol2Kit</td><td>302</td><td></td></tr><tr><td>pDEST</td><td></td><td></td><td>Gift from Geoffrey Burns labs</td></tr><tr><td>Bright field microscope&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Fluorescent microscope&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Confocal microscope</td><td></td><td></td><td></td></tr><tr><td>Image processing software</td><td></td><td></td><td></td></tr></tbody></table>

visualization of chondrocyte intercalation and directional proliferation via zebrabow clonal cell analysis in the embryonic meckel&#8217;s cartilage

脊椎动物颅面结构的发展，需要的细胞迁移，增殖，粘附和分化精确协调。该麦克尔软骨，第一咽弓衍生物的图案涉及颅神经嵴（CNC）细胞的组织分化软骨细胞的迁移和逐步分区，增殖和。期间下颚形态几项研究描述数控迁移，但的软骨细胞如何实现组织中梅克尔软骨的生长和扩展的细节仍不清楚。的SOX10限定，化学诱导Cre重组介导的重组产生截然不同的荧光蛋白（RFP，YFP和CFP）的排列，从而产生祖细胞和它们的后代的多光谱标记，反映不同的克隆群。使用共聚焦延时摄影，也能够观察到软骨细胞behavi或斑马鱼梅克尔软骨的发育过程中。 多光谱细胞标记使得科学家能够证明延伸梅克尔软骨细胞。在麦克尔软骨，这预示了下颌骨的推广阶段，软骨细胞插层，以实现扩展，因为他们在一个有组织的单细胞层排叠加。该组织嵌入过程中未能介导细胞扩展提供了蜂窝机械的解释下颌骨发育不良，我们观察下颌骨畸形。

传统的可视化软骨通过整装阿辛兰污渍是非常宝贵的观察开发麦克尔软骨和常用的可视化最终细胞组织12（ 图1A）。为了进一步分析发展中的软骨细胞加班，使用SOX10谱系追踪:枫的转基因品系，使我们能够研究细胞的迁移，融合和延伸活胚胎2,12（ 图1B）。然而，软骨细胞在颅面骨骼结构的组织仍然知之甚少。通过实时成像，所述Zebrabow-M的转?...

Watch this Scientific Journal Video about Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckelâ€™s Cartilage at JoVE.

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

颅面骨细胞组织长期以来一直推测但从未直接显现。多光谱细胞标记和体内活 成像可以在斑马鱼下颌动态细胞行为的可视化。在这里，我们详细的协议来操作Zebrabow转基因鱼，直接观察细胞嵌入和麦克尔软骨细胞的形态变化。

通过在胚胎梅克尔软骨Zebrabow克隆细胞分析软骨插层和定向扩散的可视化

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

visualization-chondrocyte-intercalation-directional-proliferation-via

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.0K 次观看.  Harvard Medical School. 颅面骨细胞组织长期以来一直推测但从未直接显现。多光谱细胞标记和体内活 成像可以在斑马鱼下颌动态细胞行为的可视化。在这里，我们详细的协议来操作Zebrabow转基因鱼，直接观察细胞嵌入和麦克尔软骨细胞的形态变化。 

视频: Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel’s Cartilage

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

观察有丝分裂和动力学在现场斑马鱼胚胎

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

科研

发育生物学

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

小鼠胚胎跖骨文化:软骨内骨化的生理模型

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Co-localization of Cell Lineage Markers and the Tomato Signal

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. Here, we used the cell lineage tracing technique to demonstrate that chondrocytes directly transform into osteoblasts and osteocytes during long bone and mandibular condyle development using two kinds of Cre, Col10a1-Cre and Aggrecan-CreERT2 (Agg-CreERT2), crossed with Rosa26tdTomato. Both Col10 and aggrecan are well-recognized markers for chondrocytes.
On this basis, we developed a new method-cell lineage tracing in conjunction with fluorescent immunohistochemistry-to define cell fate by analyzing the expression of specific cell markers. Runx2 (a marker for early-stage osteogenic cells) and Dentin matrix protein1 (DMP1; a marker for late-stage osteogenic cells) were used to identify chondrocyte-derived bone cells and their differentiation status. This combination not only broadens the application of cell lineage tracing, but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation statuses of parent cell progeny are displayed simultaneously, providing more information than cell lineage tracing alone. In conclusion, the co-application of cell lineage tracing techniques and immunofluorescence is a powerful tool for investigating cell biology in vivo.

We developed two sets of tracing combinations in Rosa26tdtomato (ubiquitously expressed in all cells)/Cre (specifically expressed in chondrocytes) mice: one with 2.3Col1a1-GFP (specific to osteoblasts) and one with immunofluorescence (specific to bone cells). The data demonstrate the direct transformation of chondrocytes into bone cells.

细胞系标记的共定位和番茄信号

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

解剖和斑马鱼的胚胎横向安装:脊髓发展分析

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

神经科学

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its longevity can be a powerful way to reveal mechanisms of tissue development and maintenance. One of the most important tools currently available to monitor cells in vivo is the Confetti mouse model. The Confetti model can be used to genetically label individual cells in living mice with various fluorescent proteins in a cell type-specific manner and monitor their fate, as well as the fate of their progeny over time, in a process called clonal genetic tracing or clonal lineage tracing. This model was generated almost a decade ago and has contributed to an improved understanding of many biological processes, particularly related to stem cell biology, development, and renewal of adult tissues. However, preserving the fluorescent signal until image collection and simultaneous capturing of various fluorescent signals is technically challenging, particularly for mineralized tissue. This publication describes a step-by-step protocol for using the Confetti model to analyze growth plate cartilage that can be applied to any mineralized or nonmineralized tissue.

This method describes the use of the R26R-Confetti (Confetti) mouse model to study mineralized tissues, covering all steps from the breeding strategy to the image acquirements. Included is a general protocol that can be applied to all soft tissues and a modified protocol that can be applied to mineralized tissues.

使用纸屑小鼠研究矿化组织的克隆遗传追踪

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

研究脊椎动物轴向伸长和分割的三维和四维可视化分析方法

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 &#176;C. 
Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

全完整型小鼠软骨外种机械负荷对软骨细胞损伤的实时可视化与分析

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

生物工程

In Vitro Preparation of Actively Maturing Bovine Articular Cartilage Explants for X-Ray Phase Contrast Imaging

Understanding the mechanisms that underpin post-natal maturation of articular cartilage is of crucial importance for designing the next generation of tissue engineering strategies and potentially repairing diseased or damaged cartilage. In general, postnatal maturation of the articular cartilage, which is a wholesale change in collagen structure and function of the tissue to accommodate growth of the organism, occurs over a timescale ranging from months to years. Conversely dissolution of the structural organization of the cartilage that also occurs over long timescales is the hallmark of tissue degeneration. Our ability to study these biological processes in detail have been enhanced by the findings that growth factors can induce precocious in vitro maturation of immature articular cartilage. The developmental and disease related changes that occur in the joint involve bone and cartilage and an ability to co-image these tissues would significantly increase our understanding of their intertwined roles.
The simultaneous visualization of soft tissue, cartilage and bone changes is nowadays a challenge to overcome for conventional preclinical imaging modalities used for the joint disease follow-up. Three-dimensional X-ray Phase-Contrast Imaging methods (PCI) have been under perpetual developments for 20 years due to high performance for imaging low density objects and their ability to provide additional information compared to conventional X-ray imaging.
In this protocol we detail the procedure used in our experiments from biopsy of the cartilage, generation of in vitro matured cartilage to data analysis of image collected using X-ray phase contrast imaging.

This protocol describes the preparation of in vitro bovine articular cartilage for imaging in high resolution with X-rays. These explants actively undergo postnatal maturation. We describe here the necessary steps from the biopsy to data analysis of 3D X-ray phase contrast imaging, passing through explant culture, tissue fixation and synchrotron preparation.

Understanding the mechanisms that underpin post-natal maturation of articular cartilage is of crucial importance for designing the next generation of ...

Optical Sectioning and Visualization of the Intervertebral Disc from Embryonic Development to Degeneration

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To decode disc degeneration and to be able to develop regenerative approaches a thorough understanding of the cellular biology of the IVD is essential. One aspect of this biology that still remains unanswered is the question of how cells are spatially arranged in a physiological state and during degeneration. The biological properties of the IVD and its availability make this tissue difficult to analyze. The present study investigates spatial chondrocyte organization in the anulus fibrosus from early embryonic development to end-stage degeneration. An optical sectioning method (Apotome) is applied to perform high resolution staining analyses using bovine embryonic tissue as an animal model and human disc tissue obtained from patients undergoing spine surgery. From a very high chondrocyte density in the early embryonic bovine disc, the number of cells decreases during gestation, growth, and maturation. In human discs, an increase in cellular density accompanied the progression of tissue degeneration. As had already been demonstrated in articular cartilage, cluster formation represents a characteristic feature of advanced disc degeneration.

We present a method to investigate spatial chondrocyte organization in the anulus fibrosus of the intervertebral disc using an optical sectioning method.

椎间盘从胚胎发育到退化的光学切片和可视化

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To ...