我们感谢加州大学洛杉矶分校高级光学显微镜/光谱实验室和加州纳米系统研究所的徕卡显微系统卓越中心（RRID:SCR_022789）提供双光子显微镜。AS得到了以色列科学基金会ISF 604-21的支持。JH 得到了 NIH/NIDCR 的R03DE030205和R01DE030471的支持。AS和JH还得到了美国-以色列两国科学基金会（BSF）的资助2021007。

所有小鼠均保存在加州大学洛杉矶分校（UCLA）或耶路撒冷希伯来大学（HUJI）的无病原体动物设施中。所有涉及小鼠的实验均根据各自机构动物护理和使用委员会 （IACUC） 批准的法规和协议进行 （ARC-2019-013;加州大学洛杉矶分校）或（MD-23-17184-3;HUJI）。实验步骤的一般工作流程如 图2A所示。有关本协议中使用的所有仪器、试剂和材料的详细信息，请参阅 材料表 。
1.溶液和凝胶的制备
<ol><li>解剖培养基:用0.5%葡萄糖制备新鲜的DMEM / F12，并在37°C下保持温暖，直到步骤2.4需要。 注意:我们使用不含酚红的 DMEM/F12 来减少实时成像期间的自发荧光。</li>
	<li>1x 培养基:使用 50% DMEM/F12、50% 大鼠血清、1x 谷氨酰胺替代品、1x MEM 非必需氨基酸、1% 葡萄糖、0.1 mg/mL L-抗坏血酸和 0.5% 青霉素-链霉素制备新鲜培养基。在37°C下保持温暖，直到步骤5.5需要为止。该培养基用于在实时成像期间培养门牙外植体。 注意:大鼠血清应是高质量的，并且专门为研究人员或商业来源的全组织培养而制备。特别是，在开始形成凝血之前，必须将血液离心（在室温下以1,200× g 离心5分钟）。离心后，应挤压所得纤维蛋白凝块并丢弃33。</li>
	<li>培养凝胶:凝胶的目的是在成像过程中固定样品，并且是新鲜制备的。<ol><li>使用微波将 200 mg 低熔点琼脂糖溶解在 10 mL DMEM/F12 中，在 DMEM/F12 中制备 2% 凝胶。将2%凝胶保持在37°C。</li>
		<li>通过混合 50% 大鼠血清、1x 谷氨酰胺替代品、1x MEM 非必需氨基酸、1% 葡萄糖、0.1 mg/mL L-抗坏血酸和 0.5% 青霉素-链霉素，制备 2x 培养基（不含 DMEM/F12）。将2x培养基（不含DMEM / F12）加热至37°C。</li>
		<li>通过混合等体积的 2% 凝胶和 2x 培养基（不含 DMEM/F12）制备 1% 培养凝胶。将1%培养凝胶保持在37°C，直到步骤5.1中需要。 注意: 混合前确保所有溶液都是温热的，以避免胶凝。我们发现 1% 凝胶适合培养成年小鼠门牙。如果要培养其他组织，应根据经验确定凝胶百分比。</li>
	</ol></li>
</ol>2.成鼠下颌骨的提取
<ol><li>使用IACUC批准的标准程序在所需年龄对小鼠实施安乐死。 注意:这里我们使用 CO2 窒息，然后是颈椎脱位。不同地区的动物安乐死法规可能有所不同。研究人员在进行实验之前应获得必要的机构批准，并确保遵守当地的动物护理法规。</li>
	<li>使用70%乙醇对小鼠进行消毒。</li>
	<li>将鼠标斩首并提取左右下颌骨。<ol><li>将鼠标放在腹部，然后使用#9单刃工业剃须刀片在颈部区域切开，并将鼠标头部与身体其他部位分开。 注意:如果还要收集咽部或颈部区域的组织，则不应进行斩首，可以直接进行步骤2.3.2。</li>
		<li>将鼠标翻转过来，使其腹侧朝上，下颌骨易于触及。</li>
		<li>用拇指和食指轻轻握住动物的头部，固定动物的头部。</li>
		<li>使用剃须刀片在矢状中段切开一个切口，从下唇到领口切开下颌皮肤。 注意:#15手术刀片也可用于切口和随后的解剖（步骤2.3.6-2.3.9）。</li>
		<li>切口时，用拇指和食指张开切开的皮肤，露出下面的肌肉和颚骨。</li>
		<li>使用剃须刀片切断下颌颊侧的咬肌，使左右半肢的外侧现在没有肌肉附着。</li>
		<li>切断下颌骨内侧的舌骨肌，以去除那里的肌肉附着物。</li>
		<li>在连接两个半骨的下颌联合处再做一个切口。切割后，下颌骨将分为左右两半。</li>
		<li>将剃须刀片楔入下颌髁和颞下颌关节之间，并小心地从头部的其余部分解剖出半侧。 注意:我们发现在切割时同时轻轻拉动门牙很有帮助。应注意避免折断下颌骨和损坏其中的软组织。</li>
	</ol></li>
	<li>立即将解剖的下颌骨转移到带有预热解剖培养基的培养皿中（步骤1.1），并使用#15手术刀片去除剩余的肌肉组织。 注意:将样品保存在冷培养基中会减慢细胞活性，导致活体成像期间某些细胞活性（如增殖或细胞运动）的回收延迟甚至失败。</li>
</ol>3.整个小鼠门牙的分离
注意:门牙的进一步分离是在明场解剖显微镜下完成的。
<ol><li>目视识别下颌骨的椭圆形区域，该区域覆盖门牙窝并容纳门牙的顶端部分。</li>
	<li>定位下颌骨，使内（舌）表面朝上。</li>
	<li>在用一对锯齿镊将下颌骨固定到位的同时，使用 #15 手术刀片沿从髁突到臼齿的方向剃掉覆盖的膜骨，在椭圆形区域生成一个窗口。这会在内表面暴露顶端切牙的软组织。</li>
	<li>将下颌骨翻转过来，使外（颊）表面朝上。</li>
	<li>如步骤3.3所述，在外下颌骨的椭圆形区域生成一个窗口，并使用手术刀的尖端挑走边缘任何剩余的骨头碎片。确保从两侧都可以看到牙齿的顶端。 注意: 剃须时避免压力过大，以防止损坏下面的软组织。</li>
	<li>系统地切掉门牙周围的骨头，以隔离整颗牙齿。<ol><li>在紧邻顶切牙的平面上做一个干净的切口，首先去除髁突。 注意: 小心不要切入门牙。</li>
		<li>在第三磨牙后方进行第二次切口，但在门牙背侧进行第二次切口，而不会损坏牙齿。这会去除包含冠状突的骨。</li>
		<li>从角突的尖端向门牙连续切割，以逐步方式逐渐去除腹侧下颌骨。 注意:牙上皮和相关的牙周组织通常粘在骨头上，如果一次切掉大部分腹骨，很容易被撕掉。为了将骨头与门牙软组织分开，可以将手术刀（或一对锋利的非锯齿镊子）插入门牙顶端的两个组织之间，然后轻轻地向前滑动器械。</li>
		<li>用臼齿切掉牙槽骨和任何仍然附着在门牙上的剩余骨头。</li>
		<li>整个门牙现在被隔离了。将完全解剖的门牙转移到具有干净的温解剖介质的培养皿中（步骤1.1）。</li>
	</ol></li>
	<li>重复步骤 3.2-3.6 以根据需要隔离其他门牙。 注意:小鼠的上颌/上门牙也维持成体干细胞，可用于研究牙齿再生34。牙科研究人员通常专注于下门牙，因为它们比上门牙更容易接近并且更容易解剖。下颌和上颌门牙祖细胞之间的差异仍有待确定。</li>
</ol>4.切除牙周组织，露出门牙上皮颈袢
<ol><li>将整个门牙放在舌侧，同时用一对锯齿镊子将牙齿固定到位，使用一对 #5 细镊子开始塞入覆盖顶端门牙和颈袢区域的牙周组织。</li>
	<li>小心地从顶芽上剥离牙周组织，使颈袢的外侧（或其他感兴趣的区域）在内窥镜下可见。 注意:应注意避免损坏牙齿上皮或将其从间充质中分离。如果门牙在感兴趣区域有荧光信号，并且有荧光解剖显微镜可用，则可以使用荧光信号来帮助区分组织类型并帮助解剖。重要的是有效地执行第 2-4 节，以便样品可以快速转移到培养基中，并通过外植体培养充分保存（见下文）。</li>
</ol>5. 用于外植体培养的组织包埋
<ol><li>将 500 μL 温热的未固化培养凝胶加入 24 孔板中的孔中，并将解剖的整个门牙快速转移到孔中。旋转板几次以冲洗门牙。</li>
	<li>将 400 μL 温热的未固化培养凝胶加入培养皿中，并将冲洗干净的门牙转移到培养皿中。 注意:我们使用与灌流设置兼容的市售培养皿。有关替代选项，请参阅步骤 6.4。</li>
	<li>定向门牙以将颈袢区域（或其他感兴趣的区域）定位在培养皿的中心（图2A，B），并调整所需成像平面的顶端切牙的倾斜度。 注意:在完全凝胶化之前，应快速进行组织定位。</li>
	<li>凝胶凝固后，使用一把细镊子去除感兴趣区域顶部的任何凝胶，使其不被凝胶覆盖。</li>
	<li>通过缓慢移液到培养皿边缘以刚好覆盖样品（~150μL）来加入足够体积的温培养基。</li>
	<li>将外植体培养物移至37°C细胞培养箱中，以使组织沉降并适应培养条件1小时。</li>
</ol>6. 门牙外植体的延时显微镜
注意:在这个实验中，我们使用了一台正置显微镜，该显微镜配备了一个数值孔径为25倍的浸水物镜。一般来说，具有高数值孔径的浸水透镜最适合深层组织成像。
<ol><li>打开显微镜和双光子激光器。</li>
	<li>将培养皿固定到载物台适配器上，并将灌注适配器环安装在顶部（图2C）。</li>
	<li>将载物台适配器连接到温度控制器集，以将培养物保持在37°C。</li>
	<li>将转接环的入口和出口连接到微灌注泵并开始灌注培养基，泵上的灌注速度设置为 20。这会在样品顶部产生缓慢流动的培养基（~5 mL/h）（图2C）。 注意: 有关将组织保持在稳定受控环境中的系统的详细信息 ，请参阅材料表 。也可以使用其他系统。也可以使用37°C加热板和自制灌注培养皿（补充图S1）的组合，该培养皿带有入口和连接到注射泵的出口。</li>
	<li>将大气控制屏障环（ACBR）放在转接环的顶部，并通过ACBR降低物镜，使其与培养基接触（图2D）。</li>
	<li>将激光波长调至 920 nm，以可视化 GFP 和红色荧光（例如 tdTomato）信号。</li>
	<li>通过目镜定位样品，然后在显微镜的软件上定位样品。</li>
	<li>使用该软件设置 Z 轴堆栈、多位置成像和时间间隔。要遵循此协议，请使用4μm的z步长和5分钟的时间间隔，持续14小时。 注意:我们发现超过5分钟的时间间隔通常不足以捕获平滑的细胞运动和分裂。</li>
	<li>启动延时成像。 注意: 样品位置可能会在第一个小时内发生变化，可能需要进一步调整。</li>
	<li>保存文件以进行下游数据处理和分析。</li>
</ol>

通过双光子显微镜实时跟踪牙齿细胞

作者没有需要披露的利益冲突。

活体组织成像是一种重要的技术，使我们能够研究细胞在其生态位环境中维持时的动态过程和行为41。理想情况下，实时成像是在 体内以高 时空分辨率进行的。然而，由于组织不可接近、光学不透明以及难以长时间固定动物或器官，哺乳动物器官的 体内 成像可能具有挑战性42。组织外植体绕过了其中一些挑战，并已在许多研究中成功采用，以跟?...

在过去的二十年中，小鼠门牙已成为研究成体干细胞调节和牙齿再生原理的宝贵平台1,2。小鼠门牙在动物的一生中不断生长并自我更新。它通过维持上皮干细胞和间充质干细胞来做到这一点，这些干细胞可以自我更新并分化成牙齿的不同细胞类型 1,2。牙科上皮干细胞产生分泌牙釉质基质的成釉细胞，而牙科间充质干细胞产生成牙细胞、牙质母细胞和成纤维细胞，分别形成牙本质、牙骨质和牙周韧带 3,4,5,6。这种新细胞的持续供应维持了组织稳态，并允许更换因咀嚼磨损或损伤而丢失的旧细胞7,8。因此，阐明调节牙科干细胞维持和分化的细胞和分子机制对于理解牙齿再生至关重要，这是一个越来越受关注的领域。
在解剖学上，成年小鼠门牙的很大一部分被包裹在颌骨中。当牙齿的切口边缘暴露时，门牙的顶端适合牙槽内，并通过牙周韧带和结缔组织牢固地附着在周围的骨骼上（图1A，B）。门牙的顶端也是牙齿的生长区域，在上皮层和间充质牙髓中维持牙干细胞和祖细胞9,10,11,12,13。具体来说，牙科上皮干细胞维持在上皮的球状末端，称为顶芽，也称为唇颈袢（图1C）。与肠上皮和表皮类似，门牙的上皮更新主要由活跃的循环干细胞及其高度增殖的中间后代（称为转运扩增细胞14,15,16,17）支持，两者都位于宫颈袢的内部。然而，门牙上皮在再生过程中是否含有和利用静止干细胞仍有待确定。相比之下，在顶端牙髓中发现了活性和静止的牙科间充质干细胞，并且静止干细胞作为储备细胞群发挥作用，在损伤修复过程中被激活13,18。
许多关于小鼠门牙更新和再生生物学的发现都是从组织学研究中获得的，其中样本在不同的时间节点获得，固定，处理，然后沿特定平面切成微米薄的切片。通过对不同小鼠模型的组织学切片进行详细分析，科学家们已经确定了不同祖细胞群的细胞谱系，以及控制门牙稳态和损伤修复的遗传和信号通路19,20,21.然而，切片中非生命细胞的静态二维 （2D） 图像无法捕捉活组织中细胞行为和空间组织的全部光谱，例如细胞形状变化、运动和细胞动力学。检测和测量这些快速的细胞变化，这些变化发生在无法通过组织切片解决的时间尺度上，需要不同的策略。此外，获取这些信息对于了解牙齿细胞如何相互作用、对不同的信号刺激做出反应以及自组织以维持组织结构和功能也至关重要。
使用双光子显微镜22 的四维 （4D） 深层组织成像的出现，是一种将三个空间维度与时间分辨率相结合的技术，通过能够对培养的组织外植体、类器官甚至原位组织进行时空检查，克服了组织学分析的固有局限性 23,24,25,26 .例如，发育中的牙齿上皮的 4D 实时成像揭示了细胞分裂和迁移的时空模式，这些模式协调组织生长、信号转导中心形成和牙齿上皮形态发生 27,28,29,30,31,32.在成年小鼠门牙中，4D成像最近被用于研究牙齿上皮损伤修复过程中的细胞行为。实时成像显示，基底上层的中间层细胞可以直接转化为基底层的成釉细胞，使受损的上皮细胞再生，挑战了上皮损伤修复的传统范式15。
在这里，我们描述了成年小鼠门牙的解剖、培养和成像，重点是唇颈袢中的上皮细胞（图 1）。该技术可保持牙细胞活力超过 12 小时，并允许以单细胞分辨率实时跟踪荧光标记的细胞。这种方法可以研究细胞运动和迁移，以及正常培养条件下细胞形状和分裂方向的动态变化，或对遗传、物理和化学扰动的反应。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24 well, flat bottom tissue culture plate</td>
			<td>Olympus plastics</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr>
			<td>25x HC IRAPO motCORR water dipping objective</td>
			<td>Leica</td>
			<td>11507704</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid (Vitamin C)</td>
			<td>Acros Organics</td>
			<td>352685000</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose bioxtra&#160;</td>
			<td>Sigma Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Delta T system&#160;</td>
			<td>Bioptechs</td>
			<td>0420-4</td>
			<td>Including temperature control, culture dishes, and perfusion setup</td>
		</tr>
		<tr>
			<td>Dissection microscope- LEICA S9E</td>
			<td>Leica</td>
			<td>LED300 SLI</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Scientific</td>
			<td>11039047</td>
			<td>Basal media without phenol red</td>
		</tr>
		<tr>
			<td>Feather surgical blade (#15)</td>
			<td>Feather</td>
			<td>72044-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>F.S.T</td>
			<td>11252-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax&#160;</td>
			<td>Thermo Scientific</td>
			<td>35050-061</td>
			<td>Glutamine substitute</td>
		</tr>
		<tr>
			<td>Leica SP8-DIVE equipped with a 25X HC IRAPO motCORR water dipping objective&#160;</td>
			<td>Leica</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>low-melting agarose</td>
			<td>NuSieve</td>
			<td>50080</td>
			<td></td>
		</tr>
		<tr>
			<td>non-essential amino acids (100x)</td>
			<td>Thermo Scientific</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin&#8211;streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15140122</td>
			<td>10,000 U/mL&#160;</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Gen Clone</td>
			<td>32-107G</td>
			<td>90 mm&#160;</td>
		</tr>
		<tr>
			<td>Rat serum</td>
			<td>Valley Biomedical</td>
			<td>AS3061SC</td>
			<td>Processed for live imaging</td>
		</tr>
		<tr>
			<td>Razor blade #9</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>F.S.T</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>F.S.T</td>
			<td>37133</td>
			<td></td>
		</tr>
		<tr>
			<td>serrated forceps</td>
			<td>F.S.T</td>
			<td>11000-13</td>
			<td></td>
		</tr>
		<tr>
			<td>spring scissors</td>
			<td>F.S.T</td>
			<td>91500-09</td>
			<td></td>
		</tr>
	</tbody>
</table>

using ex vivo live imaging to investigate cell divisions and movements during mouse dental renewal

不断生长的小鼠门牙正在成为一种高度易于处理的模型系统，用于研究成人上皮和间充质干细胞的调节以及牙齿再生。这些祖细胞群积极分裂、移动和分化，以维持组织稳态并以响应性方式再生丢失的细胞。然而，使用固定组织切片的传统分析无法捕捉细胞运动和相互作用的动态过程，限制了我们研究其调控的能力。本文描述了一种在外植体培养系统中维持整个小鼠门牙并使用多光子延时显微镜实时跟踪牙齿上皮细胞的方案。这项技术增加了我们现有的牙科研究工具箱，并允许研究人员获得有关活组织中细胞行为和组织的时空信息。我们预计这种方法将帮助研究人员进一步探索控制牙齿更新和再生过程中发生的动态细胞过程的机制。

成年小鼠门牙的顶端区域被包裹在下颌骨内（图1），因此，不能直接进入以可视化和实时跟踪居住在生长区域内的祖细胞。因此，我们开发了一种从颌骨中提取整个门牙并将其保存在外植体培养系统中进行双光子延时显微镜的方法（图2）。在这里，我们描述了捕捉上皮唇颈袢区域细胞增殖和运动的动态过程的代表性结果。
为了演...

Watch this Scientific Journal Video about Understanding Dynamic Cellular Behaviors in Adult Mouse Dental Tissue Renewal and Repairment at JoVE.com

作者聚焦:了解成年小鼠牙齿组织更新和修复中的动态细胞行为 

离体 实时成像是一种强大的技术，用于研究活组织中细胞运动和相互作用的动态过程。在这里，我们提出了一种方案，该方案实施双光子显微镜以实时跟踪培养的全成年小鼠门牙中的牙齿上皮细胞。

使用 离体 实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

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

为了在器官更新过程中保持正确的细胞组织，接近细胞的组织会移动并分裂特定的取向和模式。因此，了解这些动态细胞事件的规则和规定非常重要。小鼠门牙是成体干细胞研究的新兴模型，系统中有许多遗传工具可用于研究基因功能，并标记细胞以进行谱系追踪。迄今为止，大多数研究都使用收集的成人牙齿样本并固定其特定时间点，这阻止了我们检查生命细胞的行为。在该协议中，我们描述了一种跟踪成年小鼠门牙生活方式的方法，该技术将使未来的研究成为可能，旨在了解动态细胞行为如何有助于牙齿组织的更新和修复。

using-ex-vivo-live-imaging-to-investigate-cell-divisions-movements

Research

JoVE Journal

Developmental Biology

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

1.3K 次观看.  University of California Los Angeles. 为了在器官更新过程中保持正确的细胞组织，接近细胞的组织会移动并分裂特定的取向和模式。因此，了解这些动态细胞事件的规则和规定非常重要。小鼠门牙是成体干细胞研究的新兴模型，系统中有许多遗传工具可用于研究基因功能，并标记细胞以进行谱系追踪。迄今为止，大多数研究都使用收集的成人牙齿样本并固定其特定时间点，这阻止了我们检查生命细胞的行?...

视频: 作者聚焦:了解成年小鼠牙齿组织更新和修复中的动态细胞行为 

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

科研

发育生物学

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

运用<em&gt;离体</em&gt;全胎儿各器官的直立滴文化的过程中鼠标研究器官发育过程

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

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

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; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

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

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

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; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

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

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

使用离体实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

副本

摘要

探索更多视频

此视频中的章节

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

运用

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

分析

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

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

小鼠第二腭融合的实时成像

RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

有丝分裂过程中染色体分离的活体细胞成像

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

使用 离体 实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

副本

摘要

探索更多视频

此视频中的章节

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

运用

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

分析

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

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

小鼠第二腭融合的实时成像

RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

有丝分裂过程中染色体分离的活体细胞成像

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

使用离体实时成像研究小鼠牙齿更新过程中的细胞分裂和运动