我们要感谢阿尔瓦拉多, 他支持这项技术的初步优化和发展。在卡罗琳·阿德勒的实验室工作是由康奈尔大学开办基金支持的。

1. 准备
<ol><li>
涡水的制备17<ol>
<li>在1X 蒙锥山盐溶液中保持三角涡虫。要准备真涡虫水, 请在超纯水中制作1米CaCl2、1米MgSO4、1米氯化镁2、1米氯化钾和5米氯化钠的个人库存解决方案。过滤-消毒与0.2 µm 瓶顶 filterfor 长期储存。 注意:只使用超纯去离子水 (电阻率为 18.2 MΩ在25摄氏度), 以制备蒙锥山盐。</li>
		<li>要准备 1 L 的5X 盐溶液的股票, 结合5毫升1米 CaCl2, 5 毫升 1 M MgSO4, 0.5 毫升氯化镁2, 0.5 毫升的氯化钾和1.6 毫升的5米氯化钠溶液在900毫升超纯水。对于此解决方案, 添加 0.504 g NaHCO3并搅拌混合。用盐酸将 pH 值调整为7.0。</li>
		<li>稀释此5X 库存解决方案, 以1X 的工作浓度在无菌容器, 如大容量卡博 (见材料表) 的超纯水。使用此1X 解决方案, 以保持无性三角涡虫。 注意:作为蒙锥山盐溶液的替代品, 请使用本地购买的泉水或超纯水, 其中含有商业上可用的水族盐混合物 (参见材料表), 浓度为of0.518。</li>
		<li>为防止静态培养中的细菌感染19, 在含有抗生素的水中保持三角涡虫。在超纯水和过滤杀菌中制备硫酸庆大霉素的50毫克/毫升溶液。对于保存动物的容器, 将硫酸庆大霉素添加到最终浓度为50µg/毫升 (1:1000 稀释)。</li>
	</ol>
</li>
	<li>
肝膏的制备
	<ol>
<li>随着三角涡虫生长在一个有机的, 草喂养的牛肉肝脏的饮食, 购买新鲜的肝脏和过程中的24小时内. 取出膜胶囊, 轻轻剥离肝脏。把肝脏切成1厘米的立方体, 用刀片刮掉所有的肝静脉和动脉。</li>
		<li>浸渍肝片使用食品厂或食品处理器, 然后通过丝网食品过滤器。组合成糕点袋, 并免除注射器或35毫米培养皿。在加料前, 离心机轻轻地去除气泡。</li>
		<li>储存整除数的肝脏在-80 摄氏度, 长达一年, 并解冻之前使用。解冻后, 再冷冻任何残留的肝脏一次, 或储存在4摄氏度, 高达24小时。</li>
	</ol>
</li>
	<li>
动物的维护
	<ol>
<li>三角涡虫将生长和缩小到不同的大小取决于喂养的频率。每隔一周喂一次三角涡虫 (每14天一次)。对于长期散装文化, 请使用各种大小的塑料容器 (请参阅材料表)。</li>
		<li>为了喂养动物, 用金属铲或塑料转移吸管将豌豆大小的肝脏放在盒子里。允许动物吃1-2 小时. 在清洗盒子之前, 先取出剩余的食物。</li>
		<li>清洁蠕虫每周两次, 如果喂养 (一次直接喂养后, 两天后), 或每周一次, 如果不喂养。为了清洁, 把水倒入塑料烧杯中, 小心地浇出水, 同时保留三角涡虫在盒子里。用纸巾擦拭盒子表面, 然后在所有侧面重复, 直到盒子干净。</li>
		<li>更换水 (大约¾盒容积), 并添加庆大霉素到最后浓度的50µg/毫升。把动物存放在黑暗的柜子里或20摄氏度的孵化器里。</li>
	</ol>
</li>
	<li>
蠕虫的选择
	<ol>
<li>选择5-7 天前喂食的蠕虫。 注意:从最近喂过的蠕虫 (截肢前1-2 天) 中切除 pharynges 是非常困难的。喂食动物对叠氮化钠也更敏感。</li>
		<li>选择长度大约为6毫米的蠕虫。要估计蠕虫的大小, 请使用塑料转移吸管将其转移到培养皿中。在培养皿下面放置6英寸尺或5毫米 x 5 毫米的图形纸, 并在爬行时测量蠕虫的长度。 注意:长度小于6毫米的蠕虫更难截肢。</li>
	</ol>
</li>
	<li>
叠氮化钠的制备
	<ol>
<li>在涡水中溶解叠氮化钠粉, 制备100毫米溶液。 警告:叠氮化钠是有毒的, 应该小心处理。不要在排水管里丢弃叠氮化钠。使用后, 分开收集, 作为危险废物处置。</li>
	</ol>
</li>
</ol>2. 咽部截肢
<ol><li>将蠕虫放置在直径为35毫米的培养皿中。用塑料转移吸管小心地从盘子里取出所有的真涡虫水。 注意:最大的 20 6 毫米蠕虫可以舒适地容纳在这种大小的菜肴。</li>
	<li>将盘子放在显微镜下, 调整放大倍数, 这样可以同时查看多个蠕虫 (例如10X 放大倍数)。用100毫米叠氮化钠溶液取代真涡虫水。 注意:对于35毫米培养皿, 约5毫升叠氮化钠是足够的。解决方案应该完全淹没三角涡虫。根据需要调整大盘子的溶液体积。</li>
	<li>在显微镜下监视动物, 避免把盘子移到前3-4 分钟。</li>
	<li>通过显微镜观察咽部的挤压;咽将从咽囊中伸出并完全伸展 (大约1毫米), 如图 1B所示。 注意:重要的是要等到咽完成了完全伸展, 然后再继续。一旦咽从动物身上冒出来, 完全伸展大约需要1分钟。</li>
	<li>使用塑料转移吸管在盘子周围喷动物。把动物吸进吸管, 然后强行把它们放进盘子里几次。如果咽完全延长, 这种剧烈的吹打将导致它分离。</li>
	<li>或者, 用一对细钳 (参见材料表), 沿其长度垂直地抓住咽, 或沿其圆周水平进行, 如图 1C所示。在钳中掐咽后, 将动物向上抬向半月板。当动物被饲养时, 表面张力会导致咽部与动物分离。 注意:这种方法可以用来去除 pharynges 的小动物 (长度为1-3 毫米), 那里的强力旋转不充分。避免戳或以其他方式伤害动物的身体。</li>
	<li>使用转移吸管, 将被截肢的动物移到含有新鲜涡水的新盘子里。</li>
	<li>一旦转移, 彻底冲洗切除动物在涡水, 完全交换水三次。转移到含有涡水的新皿中, 50µg/毫升庆大霉素。 注意:在这三洗涤过程中完成缓冲交换是确保去除叠氮化钠的必要条件。</li>
	<li>第二天, 用含有50µg/毫升庆大霉素的新鲜涡水代替菜中的水。此后每隔一天重复一次。</li>
</ol>3. 截肢后咽部切除术的评估
<ol><li>检查显微镜下的动物 (用10-20X 放大) 来确定是否在成功切除咽后出现了黑点, 如图 2A所示。</li>
	<li>另外, 根据皮尔逊et ) 所描述的协议修复和漂白动物。20. 在 4 ', 6-diamidino 2-苯基吲哚 (DAPI) 或荧光共轭链亲和素 (1 毫克/毫升稀释1:500 在1X 磷酸盐缓冲盐水含0.3% 海卫 x) 中浸泡动物过夜。登上动物在 80% glycerol/20%@unit@ 磷酸盐缓冲盐水和图像在荧光显微镜下的幻灯片 (图 2B)。 注意:本手稿中的所有图像都是在一个1.0X 目标的荧光显微镜上捕获的。</li>
</ol>4. 通过测量喂养行为评估咽再生
<ol><li>
肝脏的制备
	<ol>
<li>将所需数量的肝膏转移到离心或锥形管中。简单地旋转以去除气泡。估计肝脏体积, 然后添加涡水1/5 的体积, 和2% 的总体积的红色食品着色。例如, 对1000µL 肝膏, 添加200µL 涡水和24µL 的食品着色。</li>
		<li>使用塑料杵, 吸管尖端或金属铲, 彻底混合, 直到食物着色均匀分布在肝脏膏。再旋转简要地。肝脏糊可以储存在4°c 24 小时或冷冻在整除数-80 摄氏度, 长期贮存。</li>
		<li>如果测试多达25种动物, 每道菜准备25µL 肝脏膏 (大约1µL 每只动物的肝脏糊)。</li>
	</ol>
</li>
	<li>
饲养动物
	<ol>
<li>七天后, 化学截肢, 转移动物到一个新的培养皿。如果试验10-15 只动物, 请使用35毫米菜;15多只动物应该在60毫米的培养皿中进行测试。保持动物在黑暗中, 不受干扰, 大约1小时喂养前。</li>
		<li>使用剪刀, 增加 P200 吸管尖端的宽度, 通过修剪大致½厘米关闭狭窄的末端。吸管25µL 的红肝糊入盘中。 注意:修剪末端使吹打的粘性肝糊更容易。为了防止肝脏漂浮在表面上, 当配药时, 将笔尖触到盘子的底部。</li>
		<li>允许动物喂养30分钟。</li>
		<li>通过将动物放置在白色背景上或用10-20X 放大显微镜检查它们的数量来获得吃掉的动物数目。</li>
		<li>进食后, 从盘子中取出肝脏并清洗干净。</li>
	</ol>
</li>
</ol>

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Cell Sci. 124, 228-239 (2011).</li><li>Cebri&#224;, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+nervous+system+in+the+model+planarian+Schmidtea+mediterranea:+An+immunocytochemical+study.">Organization of the nervous system in the model planarian Schmidtea mediterranea: An immunocytochemical study.</a> Neurosci. Res. 61, 375-384 (2008).</li><li>Shimoyama, S., Inoue, T., Kashima, M., Agata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Neuropeptide-Coding+Genes+Involved+in+Planarian+Pharynx+Extension.">Multiple Neuropeptide-Coding Genes Involved in Planarian Pharynx Extension.</a> Zoolog. Sci. 33, 311-319 (2016).</li><li>Ito, H., Saito, Y., Watanabe, K., Orii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epimorphic+regeneration+of+the+distal+part+of+the+planarian+pharynx.">Epimorphic regeneration of the distal part of the planarian pharynx.</a> Dev. Genes Evol. 211, 2-9 (2001).</li><li>Bueno, D., Espinosa, L., Bagu&#241;&#224;, J., Romero, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planarian+pharynx+regeneration+in++tail+fragments+monitored+with+cell-specific+monoclonal+antibodies.">Planarian pharynx regeneration in tail fragments monitored with cell-specific monoclonal antibodies.</a> Dev. Genes Evol. 206, 425-434 (1997).</li><li>Hyman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The invertebrates: Platyhelminthes and Rhynchocoela, the acoelomate Bilateria. , (1951).</li></ol>

作者没有什么可透露的。

本协议描述了用叠氮化钠选择性切除咽部的方法。三角涡虫的其他靶向消融研究使用改良手术去除感光细胞21 或药理治疗消融多巴胺能神经元22。化学截肢对现有方法的一个显著优点是它不需要手术。与真涡虫身体的其余部分相比, 咽部的刚性结构便于它完全从动物身上去除, 这是非常柔软的。此外, 咽与动物在一个小交界 (食管)30连接咽和肠道...

再生是整个动物王国发生的一种现象, 其再生能力从某些无脊椎动物的全身再生到脊椎动物的限制性能力为1。更换功能组织是一个复杂的过程, 往往需要同时恢复多种细胞类型。例如, 要再生蝾螈肢, 成骨细胞, 软骨细胞, 神经元, 肌肉和上皮细胞需要被替换为2。这些新产生的细胞类型也需要适当组织, 以促进新的肢体功能。了解这些复杂的过程需要的技术, 重点是再生的特定细胞类型和他们融入器官。
用于简化再生反应研究的策略之一是对某些细胞类型或较大的组织集合进行靶向消融。例如, 在斑马鱼中, nitroreductase 在特定细胞类型中的表达在应用甲硝唑3、4后导致其破坏。在果蝇幼虫中, 在组织特定的启动子下表达亲凋亡基因可以选择性地消融形象盘56的特定区域。这两种策略都造成了快速但受控的损伤, 并被用来解剖分子和细胞机制负责再生。
在这篇手稿中, 我们描述了一个方法, 有选择地消融整个器官称为咽在真涡虫Schmidtea mediterranea。三角涡虫是一种经典的再生模型, 以其多产的再生能力著称,其中即使是微小的碎片也能再生整个动物7, 8.它们有一个大的异构的干细胞群, 由多潜能细胞和受血统限制的祖细胞组成,9,10,11。这些细胞增殖和分化, 以取代所有缺失的组织, 包括咽, 神经, 消化系统和排泄系统, 肌肉和上皮细胞9,10,12。虽然我们知道这些干细胞开始再生, 但我们并不完全理解促使它们取代所有这些不同细胞类型的分子机制。定义的伤人方法, 使精确的干细胞反应可以帮助描绘这个复杂的过程。
咽是一个大的, 圆柱管需要喂养, 并包含神经元, 肌肉, 上皮和分泌细胞13,29。通常藏在动物腹侧的眼袋里, 在感觉到食物的存在时, 它会通过动物的单一身体开放。为了选择性地切除咽, 我们将三角涡虫浸泡在一种叫做叠氮化钠的化学物质中, 一种常用的麻醉剂,在线虫 14, 15, 16.它在三角涡虫中的使用首先由阿德勒et al 报告, 在2014年12。在接触叠氮化钠的几分钟内, 三角涡虫挤压他们的 pharynges, 并与温和的搅拌, 咽分离的动物。我们指的是这种完全和选择性的咽部 "化学截肢" 的损失。截肢后一周, 完全功能性咽部恢复为12。由于咽是需要喂养, 功能再生可以通过监测喂养行为来衡量。下面, 我们描述了化学截肢的协议, 以及评估咽部的再生和喂养行为的恢复。

<table border="0" cellpadding="0" cellspacing="0" width="1042">
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			<td height="21" style="height:21px;width:447px;">Calcium Chloride Dihydrate (CaCl2)</td>
			<td style="width:204px;">Fisher Chemical</td>
			<td style="width:155px;">C79-3</td>
			<td style="width:236px;">Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate Anhydrous (MgSO4)</td>
			<td>Fisher Chemical</td>
			<td>M65-3</td>
			<td>Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Chloride Hexahydrate (MgCl2)</td>
			<td>Acros/VWR</td>
			<td>41341-5000</td>
			<td>Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride (KCl)</td>
			<td>Acros Organics/VWR</td>
			<td>196770010</td>
			<td>Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride (NaCl)</td>
			<td>Acros Organics/VWR</td>
			<td>207790050</td>
			<td>Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Bicarbonate (NaHCO3)</td>
			<td>Acros Organics/VWR</td>
			<td>123360010</td>
			<td>Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalgene autoclavable polypropylene copolymer lowboy with spigot</td>
			<td>ThermoFisher Scientific/VWR</td>
			<td>2324-0015</td>
			<td>Storing planaria water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Instant Ocean Sea Salt</td>
			<td>Spectrum Brands</td>
			<td>Amazon</td>
			<td>Planaria water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin Sulfate</td>
			<td>Gemini Bio-products</td>
			<td>400-100P</td>
			<td>Planaria water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades</td>
			<td>Electron Microscopy Sciences</td>
			<td>71970</td>
			<td>Mincing liver&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Disposable pastry bags-16&#8221;, 12 pack</td>
			<td>Wilton</td>
			<td>Amazon</td>
			<td>Liver aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 mL syringes</td>
			<td>BD/VWR</td>
			<td>309647</td>
			<td>Liver aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes-35mm/60mm</td>
			<td>Greiner Bio-One/VWR</td>
			<td>82050-536/82050-544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic containers (various sizes)</td>
			<td>Ziploc</td>
			<td>Amazon</td>
			<td>Housing planarians in static culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipette</td>
			<td>Globe Scientific</td>
			<td>138030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps - Dumont Tweezer, Style 5</td>
			<td>Electron Microscopy Sciences&#160;</td>
			<td>72700-D (0203-5-PO)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI&#160;</td>
			<td>ThermoFisher</td>
			<td>62247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin, Alexa Fluor 488 conjugate</td>
			<td>ThermoFisher</td>
			<td>S11223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher BioReagents</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

chemical amputation and regeneration of the pharynx in the planarian schmidtea mediterranea

三角涡虫是虫非常有效的再生。他们把这种能力归功于大量的干细胞, 这些细胞能够迅速应对任何类型的损伤。在这些动物中常见的伤害模型去除大量的组织, 损害了多个器官。为了克服这种广泛的组织损伤, 我们这里描述了一种方法, 有选择地移除一个器官, 咽, 在真涡虫Schmidtea mediterranea。我们通过在含有细胞色素氧化酶抑制剂叠氮化钠的溶液中浸泡动物来实现这一目标。对叠氮化钠的短暂接触会导致咽部从动物身上挤出, 我们称之为 "化学截肢"。化学截肢除去整个咽部, 并产生一个小伤口, 咽附着在肠道。经过广泛的冲洗, 所有被截肢的动物在大约一周内再生一个完全功能的咽。身体其他部位的干细胞驱动新咽的再生。在这里, 我们提供了一个详细的化学截肢协议, 并描述了组织学和行为方法, 以评估成功的截肢和再生。

接触叠氮化钠扰乱了三角涡虫的正常运动, 导致动物伸展和扭动。这些运动迫使咽从动物腹侧出现, 在叠氮化钠溶液中大约6分钟后, 咽部的白色尖端可以看到(图 1B左面板)。几分钟后, 动物们积极地收缩, 并通过强行将咽从身体里推出来, 从而充分地伸展咽喉。(图 1B-中间面板)。在叠氮化物暴露后约11分钟,...

Watch this Scientific Journal Video about Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea at JoVE.com

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

真涡虫的化学截肢和咽部再生Schmidtea mediterranea

真涡虫Schmidtea mediterranea 是研究干细胞和组织再生的优良模型。本刊物描述一种方法, 选择性地移除一个器官, 咽, 通过暴露动物的化学叠氮化钠。本议定书还概述了监测咽部再生的方法。

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to ...

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Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

Unit 1

Regeneration and Repair

SCIENTISTS IN ACTION

10.6K 次观看.  Cornell University. 真涡虫Schmidtea mediterranea 是研究干细胞和组织再生的优良模型。本刊物描述一种方法, 选择性地移除一个器官, 咽, 通过暴露动物的化学叠氮化钠。本议定书还概述了监测咽部再生的方法。

视频: Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

的测定血细胞种群<em&gt;果蝇</em&gt;幼虫

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

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发育生物学

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

使用提顿体系的研究斑马鱼再生的分子机制

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

从斯塔利特海葵的反口Physa完全诱导再生息肉<em&gt; Nematostella vectensis</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. This morphological alteration is also recognized as epithelial to mesenchymal transition (EMT). Dysregulation of EMT is associated with fibrosis and cancer metastasis. There is emerging evidence that EMT is controlled by a number of molecular mechanisms. As such, many key genes that control apical constriction are also known to be important factors in the EMT observed in cancer metastasis. Like EMT during Drosophila gastrulation, epithelial cells can be induced to change their shape and be reprogrammed to redirect cell fate towards various other cell types. Here we provide a robust imaging method of Drosophila gastrulation to assay the initiation of morphogenetic cellular movements and cell fate identification during this stage of embryonic development. Using this method, we identify cell rearrangement at the time of gastrulation and demonstrate the importance of apical constriction during gastrulation using GFP labeled DE-cadherin.

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Herein we describe a procedure to capture live images of Drosophila gastrulation. This has enabled us to better understand the apical constriction involved in early development and further analyze mechanisms governing cellular movements during tissue structure modification.

细胞形态改变与细胞运动的影像<em&gt;果蝇</em&gt;原肠胚形成使用DE-cadherin的记者转基因果蝇

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Stentor再生的研究方法

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

成年小鼠数字截肢和再生:研究哺乳动物暴发体形成和内膜性细胞化的简单模型

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

分离大鼠脂肪组织间充质干细胞以分化为产生胰岛素的细胞

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...