这项工作的显微镜是在名古屋大学的变革性生物分子 (ITbM) 研究所进行的, 并得到日本先进植物科学网的支持。这项工作得到了日本科学技术局 (ERATO 项目到骅和 M.U.) 和日本促进科学协会的赠款的支持: 创新领域科学研究补助金 (Nos。JP24113514, JP26113710, JP15H05962, JP15H05955 M.U., Nos。JP16H06465, JP16H06464 和 JP16K21727 为 H), 补助金为年轻科学家 (B, Nos。JP24770045 和 JP26840093 为 M.U.), 和补助金挑战探索性研究 (No。JP16K14753 为 M.U.)。

1. 制备 体外 胚珠培养基 <ol> <li> 为 体外培养的液体培养基 (和 #34; N5T 培养基和 #34;) 含有 1x Nitsch 基盐混合物, 5% (w/v) 海藻糖二水合物, 0.05% (w/v) 2-(n-吗) 磺酸 (MES)-KOH (pH 5.8) 和 1x Gamborg 和 #39; s 维生素溶液. </li>
	<li> 使用 KOH 调整 pH 值为 5.8. </li>
	<li> 通过灭菌 (121 和 #176; C, 20 min) 对介质进行消毒, 或者通过0.22 和 #181; m 过滤器过滤. 
	注: 灭菌介质可存储在4和 #176; C 为 2-3 月. </li>
</ol> 2。Micropillar 阵列设备的制备 <ol> <li> 用刀、刀片或剪刀将 Micropillar 设备切割成玻璃部分 (14 毫米和 #966;) 35 mm 的玻璃底盘. 
	注: 在以前的论文 8 , 9 中描述了该设备构造的完整过程, 因此此处省略了详细信息。玻璃底盘的体积较小, 如4井或96井板, 可用于短期成像而不使用该设备. </li>
	<li> 在紫外线照射下, 在15分钟内对硅橡胶设备的上部进行消毒. </li>
	<li> 使用方形尖端镊子将该设备翻转过来, 并将其置于 UV 光下15分钟以消毒底部. </li>
	<li> 将灭菌的硅橡胶设备转移到 35 mm 培养皿中, 添加 N5T 介质, 直到设备完全浸泡在介质中 (约 5-7 毫升). </li>
	<li> 把盘子放进真空室, 减少对德加的压力。将吸尘器保持3小时至一夜, 直到设备中的空气被介质取代. 
	注: 该装置可在强真空下通过气泡分离. </li>
	<li> 将该设备从盘中取出, 用方形尖端镊子将其放在纸巾上, 将多余的介质放在侧面和底部. 
	注意: micropillar 阵列上的介质 (上表面) 不应被移除, 因为这将用于胚珠的培养. </li>
	<li> 将设备传输到 76 mm x 26 mm 的滑动玻璃上, 确保将 micropillar 部分保持为上部, 并将设备与 35 mm 培养皿盖盖在一起, 以防止在下列胚珠萃取过程中干燥. </li>
</ol> 3。丝解剖和胚珠提取 <ol> <li> 用70% 乙醇擦拭针 (0.40 mm) 和细镊子。针很容易通过将它附着在注射器或木棍上来处理. </li>
	<li> 检查植物的花序, 并选择合适的角果进行实验。大约5毫米角果包含受精卵, 并且 8-10 毫米角果包括幼小球状胚胎. </li>
	<li> 使用镊子将角果, 并将其放在 76 x 26 mm 滑动玻璃上的双面胶带上。一个丝包含约 40-60 胚珠, 和 3-4 角果 ( 即 , 120-240 胚珠) 是足够的一个设备. </li>
	<li> 打开丝 (子房壁) 以在显微镜下使用灭菌针和镊子查看胚珠。仅切割子房壁, 使胚珠未损坏. </li>
	<li> 将打开的丝转到 N5T 的设备上 (在步骤2.7 中准备), 然后用针或镊子将胚珠释放到介质中. </li>
	<li> 将一个小盖玻璃 (18 mm x 18 mm) 放到该设备上, 将胚珠推入 micropillar 阵列中的空间. </li>
	<li> 通过用镊子或手指水平拉动盖子玻璃以去除多余的介质. </li>
	<li> 将该设备倒置, 将其放入 35 mm 的玻璃底盘中, 并通过使用方形尖端镊子将其粘贴到玻璃上, 轻轻按压该设备. </li>
	<li> 将 N5T 介质轻轻地倒入玻璃底盘中, 迁中瓶, 直到将其完全浸泡在介质中, 然后用石蜡膜封住盘. 
	注: 该设备可以回收, 但太旧的设备很容易脱离玻璃底部。此外, 如果在步骤2.5 中未完全脱, 则该设备可能会浮动, 或者介质被浇得太快. </li>
</ol> <img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55975/55975fig2.jpg"/> 图 2: 示例准备的示意图程序. 
此示意图流对应于步骤3.5 到4.1。<a href="//ecsource.jove.com/files/ftp_upload/55975/55975fig2large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
 4. 延时成像 <ol> <li> 将在步骤3.9 中准备的玻璃底盘放在倒置显微镜下, 并选择合适的胚珠来聚焦。
	注意: 由于胚珠在舞台、位置和方向上各不相同, 因此应通过检查其标记荧光来找到合适的胚珠. </li>
	<li> 根据显微镜系统的制造商和 #39 的指示启动实时成像. 
	注意: 显微镜设备和参数是多种多样的, 因此用户应该选择适合实验目的的合适设置。例如, 此手稿中使用的设置在 表 1 中列出. </li>
</ol> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"> <tbody> <tr> <td> 设备/设置 </td> <td> 图 3 (A) 和补充视频1和 4 </td> <td>图 3 (B) 和补充视频 2 </td> <td> 辅助视频 3 </td> </tr> <tr> 显微镜 <td> </td> 激光扫描倒置显微镜 (A1R MP) <td> </td> 旋转盘共聚焦倒置显微镜 (CSU-W1) <td> </td>箱式倒置共焦显微镜系统具有稳定的孵化室 (CV1000) </td> </tr> <tr> <td> 激光 </td> <td> 钛: 蓝宝石飞秒脉冲激光 </td> <td> 488 nm 和 561 nm ld 激光器 </td> <td> 488 nm 和 561 nm ld 激光器 </td> </tr> <tr> <td> O目的透镜和 #160; </td> <td> 40X 浸水物镜 (na = 1.15) 与浸没介质 </td> <td> 60X 硅油浸泡物镜 (na = 1.30), 安装在压电聚焦驱动器 </td> <td> 40X 物镜 (na = 0.95) </td> </tr> <tr> <td> 检测或 </td> <td> 外部 non-descanned GaAsP PMT 探测器 </td> <td> EMCCD 摄像机 </td> <td> EMCCD 摄像机 </td> </tr> <tr> <td> 分色镜像 </td> <td> DM495 和 DM560 </td> <td> DM488/561 </td> <td> DM400-410/488/561 </td> </tr> <tr> 过滤器 <td>> 带通滤波器;534/30 nm 和 578/105 nm </td> <td> 带通滤波器; 520/35 nm 和 593/46 nm </td> <td> 带通滤波器; 520/50 nm 和 617/73 nm </td> </tr> <tr> <td> 切片沿 z-axis </td> <td> 31 z 堆栈与1和 #181; m 间隔时间 </td> <td> 17 z 堆栈与3和 #181; m 间vals </td> <td> 7 z 堆栈, 具有5和 #181; m 间隔时间 </td> </tr> <tr> <td> 时间间隔 </td> <td> 20 分钟 </td> <td> 5 分钟 </td> <td> 10 分钟 </td> </tr> </tbody> </table> 表 1:本手稿中使用的显微镜系统和设置. 
显微镜和参数是多种多样的, 因此每个用户都应该选择合适的实验系统. 
<ol start="3"> <li> 检查获取的图像序列, 并将其转换为常规电影格式, 如. avi 或。
	注意: 如果显微镜软件无法将图像输出为电影格式, 则可以将图像保存为. tif, 然后在 ImageJ 中打开它们, 这是一个由 NIH 图像启发的开源程序, 用于更改文件类型。ImageJ 提供: https://imagej.nih.gov/ij/。ImageJ 也可以用来做一个 Z s塔克电影, 如最大强度投影, 如所述: https://imagej.net/Z-functions. </li>
</ol>

<ol>
	<li>Natesh, S., Rau, M. A., Johri, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Embryology of Angiosperms. , 377-443 (1984).</li><li>Kranz, E., Lorz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+fertilisation+of+maize+by+single+egg+and+sperm+cell+protoplast+fusion+mediated+by+high+calcium+and+high+pH.">In vitro fertilisation of maize by single egg and sperm cell protoplast fusion mediated by high calcium and high pH.</a> Zygote. 2 (2), 125-128 (1994).</li><li>Uchiumi, T., Uemura, I., Okamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+in+vitro+fertilization+system+in+rice+(Oryza+sativa+L.).">Establishment of an in vitro fertilization system in rice (Oryza sativa L.).</a> Planta. 226 (3), 581-589 (2007).</li><li>Sun, M. X., Moscatelli, A., Yang, H. Y., Cresti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+double+fertilization+in+Nicotiana+tabacum+(L.):+the+role+of+cell+volume+in+cell+fusion.">In vitro double fertilization in Nicotiana tabacum (L.): the role of cell volume in cell fusion.</a> Sex Plant Rep. 13 (4), 225-229 (2001).</li><li>Tian, H. Q., Yuan, T., Russell, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+double+fertilization+and+the+cell+cycle+in+male+and+female+gametes+of+tobacco.">Relationship between double fertilization and the cell cycle in male and female gametes of tobacco.</a> Sex Plant Rep. 17 (5), 243-252 (2004).</li><li>Costa, L. 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=Central+cell-derived+peptides+regulate+early+embryo+patterning+in+flowering+plants.">Central cell-derived peptides regulate early embryo patterning in flowering plants.</a> Science. 344 (6180), 168-172 (2014).</li><li>Sauer, M., Friml, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+of+Arabidopsis+embryos+within+their+ovules.">In vitro culture of Arabidopsis embryos within their ovules.</a> Plant J. 40 (5), 835-843 (2004).</li><li>Gooh, K., 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=Live-cell+imaging+and+optical+manipulation+of+Arabidopsis+early+embryogenesis.">Live-cell imaging and optical manipulation of Arabidopsis early embryogenesis.</a> Dev Cell. 34 (2), 242-251 (2015).</li><li>Park, J., Kurihara, D., Higashiyama, T., Arata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+microcage+arrays+to+fix+plant+ovules+for+long-term+live+imaging+and+observation.">Fabrication of microcage arrays to fix plant ovules for long-term live imaging and observation.</a> Sens Act B-Chem. 191, 178-185 (2014).</li><li>Kimata, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeleton+dynamics+control+the+first+asymmetric+cell+division+in+Arabidopsis+zygote.">Cytoskeleton dynamics control the first asymmetric cell division in Arabidopsis zygote.</a> Proc Natl Acad Sci U S A. 113 (49), 14157-14162 (2016).</li><li>Nambo, 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=Combination+of+Synthetic+Chemistry+and+Live-Cell+Imaging+Identified+a+Rapid+Cell+Division+Inhibitor+in+Tobacco+and+Arabidopsis+thaliana.">Combination of Synthetic Chemistry and Live-Cell Imaging Identified a Rapid Cell Division Inhibitor in Tobacco and Arabidopsis thaliana.</a> Plant Cell Physiol. 57 (11), 2255-2268 (2016).</li><li>Mizuta, Y., Kurihara, D., Higashiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+with+longer+wavelength+excitation+in+intact+Arabidopsis+tissues.">Two-photon imaging with longer wavelength excitation in intact Arabidopsis tissues.</a> Protoplasma. 252 (5), 1231-1240 (2015).</li></ol>

作者没有什么可透露的。

这份手稿介绍了一个简单的体外胚珠培养协议, 这是有效的使用在活细胞成像的发展受精卵和胚胎。
根据胚阶段的需要, 对该装置的设计进行优化。第一个被开发的设备是一个 microcage 阵列调整方向和固定胚珠9的位置, 然后构造一个 micropillar 设备来更有效地捕获胚珠8。此外, micropillars 比 microcage 更薄、更柔软, 因此不会在长期观察中?...

生物体的基本身体计划由单细胞受精卵发育而来。在大多数开花植物中, 合分裂产生一个顶端和一个基底细胞, 分别发育成芽和根,1。因此, 了解植物体在胚胎发生过程中是如何形成的是很重要的, 但是没有一个有效的工具可以直接观察受精卵和胚胎的动态, 因为它们在花朵深处发育。在一些单子叶植物种, 如玉米和大米, 一个体外受精方法已经建立2,3。在这种方法中, 分离的精子和卵细胞被电或化学熔化, 生成的细胞可以发展成一个肥沃的植物。然而, 在双子植物, 没有体外受精方法, 可以产生适当的胚胎, 大概是由于同步细胞周期状态的男性和女性的配子4,5。此外, 胚周围组织 (胚乳) 在胚胎发育中起重要作用6。
在一个模型双子物种,一. 南芥, 一个体外培养方法是通过专注于整个胚珠, 其中包含胚胎和胚乳的7。该系统成功地用于分析各种化学试剂对胚胎发生的影响, 但由于其存活率低, 不宜用于延时成像。因此, 开发了一种新的体外胚珠培养体系, 以便早于合子阶段开始, 并在高比例的8中产生肥沃的植株。经多次试验, 发现 Nitsch 培养基和海藻糖对胚珠的存活率有显著提高8。此外, 由于胚珠随着它的生长而扩展, 因此经常从显微镜的观察场移开, 因此开发了一种用于在培养基9中固定胚珠的硅橡胶装置。该设备使长期成像 3-4 天, 这是足够的跟踪发展从受精卵到心脏阶段的胚胎。使用这种方法, 不仅在正常条件下, 而且在化学抑制剂或各种突变体背景下, 也可以可视化受精卵极化和胚模式的动态,8,10 ,11。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55975/55975fig1.jpg"> 
图 1: 用于显示受精卵和胚胎通过胚珠的特定荧光标记的原理图。 拟南芥受精卵发育成胚珠中的胚, 这是在花深处产生的。在这个体外培养系统中, 通过胚珠观察受精卵和胚胎, 因此使用其他胚珠组织中没有表达的特定荧光标记是很重要的。 <a href="//ecsource.jove.com/files/ftp_upload/55975/55975fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table><tbody><tr><td>Nitsch basal salt mixture</td><td>Duchefa</td><td>N0223</td><td></td></tr><tr><td>trehalose dihydrate</td><td>Wako Pure Chemical</td><td>206-18455</td><td></td></tr><tr><td>MES</td><td>Dojindo</td><td>345-01625</td><td></td></tr><tr><td>Gamborg&amp;rsquo;s vitamin solution</td><td>Sigma-Aldrich</td><td>G1019</td><td></td></tr><tr><td>35-mm glass-bottom dish</td><td>Matsunami Glass</td><td>D111300</td><td></td></tr><tr><td>35 mm culture dish</td><td>Corning</td><td>430588</td><td></td></tr><tr><td>PDMS</td><td>Dow Corning Co.</td><td>Sylgard184</td><td></td></tr><tr><td>76 &amp;times; 26 mm slide glass</td><td>Matsunami Glass</td><td>S1225</td><td></td></tr><tr><td>18 &amp;times; 18 mm slide glass</td><td>Matsunami Glass</td><td>C018181</td><td></td></tr><tr><td>needle (gauge 0.40 mm)</td><td>Terumo</td><td>NN-2719S</td><td></td></tr><tr><td>Immersion medium Immersol W 2010</td><td>Zeiss</td><td>444969-0000-000</td><td></td></tr><tr><td>A1R MP</td><td>Nikon</td><td>A1RsiMP(1080) Ti-E-TIRF</td><td></td></tr><tr><td>CSU-W1</td><td>Yokogawa Electric</td><td></td><td>It is a customized equipment, and thus Catalog Number is not avairable.</td></tr><tr><td>CV1000</td><td>Yokogawa Electric</td><td>CV1000-SP84</td><td></td></tr></tbody></table>

in vitro ovule cultivation for live-cell imaging of zygote polarization and embryo patterning in arabidopsis thaliana

在大多数开花植物中, 受精卵和胚都深藏在母体组织中, 因此长期以来一直是它们动态发展的奥秘;例如, 受精卵对立如何建立体轴, 以及胚胎在器官形成过程中如何指定各种细胞的命运。这份手稿描述了一个体外胚珠培养方法, 以执行受精卵和胚胎的活体细胞成像的拟南芥。优化的培养基允许受精卵或早期胚胎长成肥沃的植物。将其与聚 (烷) micropillar 阵列器件相结合, 在同一位置的液体介质中保存胚珠。这种固定是至关重要的观察相同的胚珠在显微镜下数天, 从合分裂到晚期胚胎阶段。由此产生的活细胞成像可用于监测受精卵极化的 real-time 动力学, 如细胞核迁移和细胞骨架重排, 以及胚胎模式中的细胞分裂时序和电池命运规范。此外, 这种胚珠培养系统可以与抑制剂结合, 分析各种因素对胚胎发育的影响, 并与光学操作, 如激光干扰, 以检查细胞通讯的作用。

利用这种胚珠培养体系, 可以跟踪受精卵极化和胚模式的生存动力学。这是一个成就, 因为以前没有技术来可视化的 real-time 行为的受精卵和胚胎, 这是隐藏在母亲的组织深处。图 3A和补充视频 1显示年轻合子中的微管 (MTs) 在尖端区域10中累计为一个横向环。这 MT 圆环被维护, 当受精卵拉长在顶端方向。在完成合子伸长和?...

Watch this Scientific Journal Video about In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana at JoVE.com

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

这份手稿描述了一个体外胚珠培养方法, 使受精卵和胚胎的活细胞成像的拟南芥。该方法用于在受精卵分化过程中对细胞内动力学进行可视化, 并对发育中的胚胎的胞命运规范进行研究。

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

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Developmental Biology

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

10.8K 次观看.  Nagoya University. 这份手稿描述了一个体外胚珠培养方法, 使受精卵和胚胎的活细胞成像的拟南芥。该方法用于在受精卵分化过程中对细胞内动力学进行可视化, 并对发育中的胚胎的胞命运规范进行研究。

视频: In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

协议获得合子和体细胞胚胎研究早期胚胎发育的调控中的示范豆科<em&gt;苜蓿</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

科研

发育生物学

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Geomagnetic Field (Gmf) and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) and the moment of the radiation of Angiosperms. This led to the hypothesis that alterations in GMF polarity may play a role in plant evolution. Here, we describe a method to test this hypothesis by exposing Arabidopsis thaliana to artificially reversed GMF conditions. We used a three-axis magnetometer and the collected data were used to calculate the magnitude of the GMF. Three DC power supplies were connected to three Helmholtz coil pairs and were controlled by a computer to alter the GMF conditions. Plants grown in Petri plates were exposed to both normal and reversed GMF conditions. Sham exposure experiments were also performed. Exposed plants were photographed during the experiment and images were analyzed to calculate root length and leaf areas. Arabidopsis total RNA was extracted and Quantitative Real Time-PCR (qPCR) analyses were performed on gene expression of CRUCIFERIN 3 (CRU3), copper transport protein1 (COTP1), Redox Responsive Transcription Factor1 (RRTF1), Fe Superoxide Dismutase 1, (FSD1), Catalase3 (CAT3), Thylakoidal Ascorbate Peroxidase (TAPX), a cytosolic Ascorbate Peroxidase1 (APX1), and NADPH/respiratory burst oxidase protein D (RbohD). Four different reference genes were analysed to normalize the results of the qPCR. The best of the four genes was selected and the most stable gene for normalization was used. Our data show for the first time that reversing the GMF polarity using triaxial coils has significant effects on plant growth and gene expression. This supports the hypothesis that GMF reversal contributes to inducing changes in plant development that might justify a higher selective pressure, eventually leading to plant evolution.

Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

This protocol describes a method to test the hypothesis that the reversal of the effective geomagnetic field (GMF) induces differential gene expression and alters the morphology of Arabidopsis thaliana. A triaxial octagonal system of Helmholtz coil-pairs was used to artificially generate reversed GMF conditions in the plant growth chamber.

地磁场（GMF）和植物进化:调查逆转基因食品对影响<em&gt;拟南芥</em&gt;开发与基因表达

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) ...

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

住发展拟南芥花的共焦成像

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

A Method for Characterizing Embryogenesis in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

表征在拟南芥胚方法

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes&#8217; nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

We demonstrate a method to determine successful or failed fertilization on the basis of sperm nuclear morphology in Arabidopsis double fertilization using an epifluorescence microscope.

通过追踪阿拉伯植物双重受精中的精子核形态对受精国的评价

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

生物学

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female gametophyte are essential for double fertilization and subsequent seed development. The interactions between male and female gametophytes during pollen tube reception culminate in pollen tube rupture and the release of two sperm cells to effect double fertilization. As pollen tube growth and double fertilization are deeply hidden within the tissues of the flower, this process is difficult to observe in vivo.
A semi-in vitro (SIV) method for the live-cell imaging of fertilization in the model plant Arabidopsis thaliana has been developed and implemented in several investigations. These studies have helped to elucidate the fundamental features of how the fertilization process occurs in flowering plants and which cellular and molecular changes occur during the interaction of the male and female gametophytes. However, because these live cell imaging experiments involve the excision of individual ovules, they are limited to a low number of observations per imaging session, making this approach tedious and very time-consuming. Among other technical difficulties, a failure of the pollen tubes to fertilize the ovules in vitro is&#160;often reported, which severely confounds such analyses.
Here, a detailed video protocol for the imaging of pollen tube reception and fertilization in an automated and high-throughput manner is provided, allowing for up to 40 observations of pollen tube reception and rupture per imaging session. Coupled with the use of genetically encoded biosensors and marker lines, this method enables the generation of large sample sizes with a reduced time investment. Nuances and critical points of the technique, including flower staging, dissection, medium preparation, and imaging, are clearly detailed in video format to facilitate future research on the dynamics of pollen tube guidance, reception, and double fertilization.

Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method

Here, we describe an improvement of the semi-in vitro (SIV) method for observing pollen tube guidance and reception in Arabidopsis thaliana,&#160;which increases the receptivity of ovules. The high-throughput SIV cum septum method may be coupled with gametophyte marker lines and genetically encoded biosensors to monitor the dynamic process of fertilization.

拟 南芥 花粉管接收和半体外隔膜法 双受精的实时成像

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female ...

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

摘要

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此视频中的章节

协议获得合子和体细胞胚胎研究早期胚胎发育的调控中的示范豆科

胸腺表征祖定居在小鼠胚胎使用

地磁场（GMF）和植物进化:调查逆转基因食品对影响

住发展拟南芥花的共焦成像

表征在拟南芥胚方法

通过追踪阿拉伯植物双重受精中的精子核形态对受精国的评价

从长期来看，高解析度激光共聚焦时间流逝成像

高效，快速的早期胚胎分离

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法

拟南芥花粉管接收和半体外隔膜法双受精的实时成像

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

摘要

探索更多视频

此视频中的章节

协议获得合子和体细胞胚胎研究早期胚胎发育的调控中的示范豆科

胸腺表征祖定居在小鼠胚胎使用

地磁场（GMF）和植物进化:调查逆转基因食品对影响

住发展拟南芥花的共焦成像

表征在拟南芥胚方法

通过追踪阿拉伯植物双重受精中的精子核形态对受精国的评价

从长期来看，高解析度激光共聚焦时间流逝成像

高效，快速的早期胚胎分离

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法

拟 南芥 花粉管接收和半体外隔膜法 双受精的实时成像

拟南芥花粉管接收和半体外隔膜法双受精的实时成像