这项工作是由美国国立卫生研究院资助DP2OD008470支持。

该程序与本协议描述的斑马鱼胚胎工作在Notre Dame大学被批准的机构动物护理和使用委员会。 1.林格氏液的制备<ol><li>使116 mM氯化钠，2.9 mM的氯化钾，3.6毫米氯化钙2和5毫摩尔的Hepes用超纯的H 2ö的溶液通过连续搅拌，以避免任何盐沉淀。 </li><li>加入盐和溶液的同时仍然搅拌后，加入100单位/青霉素每毫升每毫升100微克链霉素。 </li><li>加入所有抗生素和盐后，执行在无菌容器中的溶液和存储的过滤器灭菌。 注意：N-二苯基硫脲（1-苯基-2-硫脲）或PTU可加入林格氏液来阻止颜料在胎圈植入胚胎的形成没有负影响到胚胎健康。为了准备林格/色素沉着封闭液，使用concentration 0.003％机动部队。由于PTU的低浓度添加它是表示，林格氏液的较大体积进行，至少1升，以避免增加PTU粉末的微乎其微的量。在步骤1.2添加PTU，而解决方案是震撼人心。 </li></ol> 2.三卡因溶液制备<ol><li>加入2克乙基3-氨基苯甲酸甲酸盐（三卡因）每升的溶液加0.1摩尔Tris，从1M的股票至pH 9.5平衡的和用超纯H 2 O制成 注意：三卡因将用于安乐死的斑马鱼胚胎在期望的时间点，以测定珠子植入表型。 </li></ol> 3. E3溶液的制备<ol><li>使0.25 M氯化钠，的10mM氯化钾，12.5毫米氯化钙2，和16.6毫硫酸镁至1升超纯水的溶液来创建1升的E3。 </li><li>加入200μl亚甲基蓝的E3解决方案来抑制真菌生长。 </li></ol>为微珠转移4.牵引针<ol><li>用火抛光的硼硅玻璃长丝在一个外径：1.0mm时，编号：0.5毫米，在10厘米的长度。 </li><li>将borosilicated玻璃成微量拉马准备细针。 注意：以下四个方面可作为一个起点，以拉动针：热= 540，拉= 245，速度= 200，和时间= 125。 </li><li>后拉动针的足够数量的，存储在有盖的塑料陪替氏培养皿，放置在橡皮泥或胶带，以防止破坏针尖的条，直至使用。 </li><li>修剪针尖到所需的缸径，用一双细镊子将比分针的结束。 注意：针孔大小将取决于所利用的微珠大小以及用户操控的喜好而变化。一个很好的出发点是工艺孔的尺寸在100至200微米的范围内，如果微珠从50-100微米会被利用。 </li><li> PRIOR来使用，将修剪针插入毛细管持有人的方式完成转让微珠仪器。 </li></ol> 5.晶须/睫毛工具准备<ol><li>获取晶须，睫毛，或其他小而坚定的一块天然或合成的头发丝。切断丝的根部附近，在一个45°角。坚持晶须使用永久性透明胶胶水P-1000枪头。 </li></ol> 6.微珠植入托盘准备<ol><li>混合2克琼脂糖用100ml的E3溶液进行2％的E3琼脂糖凝胶。 </li><li>加热1分钟和30秒的2％琼脂糖溶液在250毫升的Erlenmeyer烧瓶中，旋动烧瓶，每30秒，以避免从鼓泡过的凝胶。 </li><li>取60毫米×15毫米的培养皿的盖子，将其放置在一个较大的150毫米×15毫米培养皿与内部朝上。 </li><li>倒入热的琼脂糖凝胶为150毫米×15陪替氏培养皿，并确保固定下来的小培养皿用食指盖子。 注：允许一薄层的小培养皿盖子下面琼脂糖以除去缩合，将形成浇注的凝胶时;这将使珠更容易解剖显微镜下才能看到。 </li><li>如琼脂糖冷却，但其固化​​之前，放置一个井模具上的凝胶。这将允许在胚胎更容易放置。 </li><li>注意：为了避免在孔中的气泡，慢慢将模具成一角度，直到其浮在凝胶的顶部。 </li><li>使用microspatula，小心地取出模具以及一旦凝胶凝固。加E3溶液，并储存于4℃。 </li></ol> 7.胚胎收集<ol><li>准备交配室，由隔板隔开，通过填写他们与制水，然后把成年斑马鱼的男性（S）和女性（S）对在室O / N。 </li><li>收集用细铁丝网过滤受精卵（胚胎阶段会略有不同）。 </li><li>通过转动过滤器倒置并与E3的微细流漂洗它存入受精卵在一个干净的，直径10厘米的培养皿中，直到没有鸡蛋留在过滤器，并有大约25毫升的菜E3溶液。 </li><li>采集后，将划分成鸡蛋几盘（每盘约50-60蛋），以避免过度拥挤，这可能会导致发育不同步离合器内，并收集所需的时间点很难。 </li><li>孵育在E3溶液胚胎在28.5℃下根据标准斑马鱼分段系列40，使发展的评估。 注意：胚胎应转移到E3 / PTU在24小时受精后（HPF），以防止颜料的发展，如果操作在年龄较大的阶段必要的光学清晰度。 </li></ol> 8.植入珠<ol><li>孵育在所希望的测试分子浓度的微珠或相应的车辆solution用于在适当的体积所需的时间。 注意：研究者可以使用的无菌培养皿中，从3-6厘米，直径，或多孔板，这两者使所述溶液中的微珠的可视化。时间可根据类型和感兴趣的分子的量变化，并且研究者应参考制造商或发表的建议为孵化和其它方面的持续时间，例如畏光和温度。 </li><li>一旦胚胎已达到所希望的发育时间点，从使用细镊子其绒毛膜取出胚胎。 </li><li>孵育在用抗生素进行10分钟的过滤林格氏溶液胚胎将它们适应到该溶液中。 </li><li>而将胚驯化，转移微珠进微珠板注入托盘内坐落和冲洗他们在林格氏溶液10分钟。 注：达到该点之后植入物中50分钟的珠子。该W病减少，一个将植入与感兴趣的分子的较小浓度的珠子的可能性。 </li><li>阵列胚胎成微珠注入托盘的孔中，注意到定向它们，以便感兴趣的区域，其中将植入的微珠可访问。 </li><li>请使用钨针胚胎小切口。 </li><li>转移微珠上使用微毛细管移液管胚胎：按压吸管的灯泡，轻轻拉动微珠到毛细管柱，然后释放到在期望的目的地传送所述微珠中的压力。 </li><li>使用晶须/睫毛工具来定位胚胎内的微珠。注意：一定要微珠从切口部位插入定位组织这样走微珠不会从胚胎排出，因为它愈合。 </li></ol>

The authors have nothing to disclose.

在过去的一个世纪中，车身图案计划和器官的认识已经发生了巨大的进步。组织操纵技术是在揭露这些生命过程的关键信息是至关重要的。遗传修饰是最常用的方法为确定基因功能之一，以及镶嵌分析的方法，如细胞移植，提供了有用的方法来了解基因功能的自治。微珠植入提供了另外一个场地来询问特定的分子如何改变发展动态，因为这种方法可以使研究者通过引入信号分子或抑制剂改变局部组织环境。不同微珠的范围是市售的，即具有不同的尺寸和其它物理特性存在（ 例如，电荷），使得它们可以用于感兴趣的实验条件。因此，通过注入被浸泡在蛋白质或CHEMI微珠的生物体内的感兴趣校准，研究人员可以调查发展过程中的局部效果并找到基因或与特定生物的表型（多个）分子之间的关联。 的研究，例如由和田执行所述一个和同事使用微珠注入，以评估在斑马鱼23增加Hedgehog信号的骨骼图案形成于前脑颅（ANC）的效果。以前的研究已经表明，Shh的需要的ANC形成14。使用嘘涂层微珠研究人员发现，这种信号促进软骨形成的ANC。微珠植入过程中用来证明在ANC Hedgehog信号和软骨的形成之间的明确联系。在斑马鱼这一组织操纵技术的另一个重要的例子是观察研究中，科学家调查了红细胞瘤二十六的转录控制（ETS）域˚F演员ETS相关分子（ERM）及多瘤病毒增强激活3（PEA3）由FGF信号在斑马鱼早期大脑发育19。通过微珠注入实验中，他们能够表明，FGF8和FGF3可以异位激活嗯和PEA3的表达。这些实施例说明微珠的实用程序来提供深入了解在组织形成互相协作的发展机制，其可以充分表征通过使用方法来评估基因表达41。因此，微珠移植可以是一个可行的方法探索其他组织，如中间胚层（IM），其产生于肾脏。具体而言，这将有助于肾脏发育研究中，研究分子如何影响肾单位分割42和小管43,44，这只是表面上理解目前的进程。此外，微珠注入已经开始被用于螺柱在斑马鱼29]和y再生过程可适于用于与任意数量的器官再生模式，如像肾单位45或结合的方法已被配制成与相应的成人结构进行的研究，胚胎组织的以下激光烧蚀的使用46-49。最后，微珠植入在人类疾病的模型，如癌症50,51或组织变性52,53中使用的潜力。 在本协议中，我们证明了微珠植入在斑马鱼胚胎，这也由其他研究人员已经类似地描述，但通过视频协议1中未示出的方法。以最小的实践中，我们能够在植入胚胎8-10的近似率/小时，其中涉及此过程的可行性，一旦研究人员有一定的经验微珠。本文所示的结果表明，珠的各种ðimensions可以在早期阶段被植入，并且小心，这个组织操纵技术可以与对胚胎最小物理破坏来实现。应当强调的一个改进是使用了晶须/睫毛工具微珠定位成胚胎。这种相对便宜和简单的设备是大约相同的直径作为微珠，容易得到，并有助于加快注入工艺。晶须/间隙可切割成所希望的长度，以产生一个坚固而有微妙的珠处理工具，这取决于研究者灵巧和偏好。最后，虽然我们在这里如何物理操纵微珠斑马鱼胚胎着床进行描述，这个协议没有具体的轮廓处理程序不同的药物或肽。在一般情况下，经化学处理的微珠应注入动物提供权宜之计，以避免在有机体的其它区域不希望的影响，和研究人员应清楚在关于形成与他们开始研究之前任何此类化学品有关的可能的安全问题。 总之，我们已经证明了一个相对简单的和有效的微珠注入法具有广泛的使用材料是在实验室中容易获得的应用程序。最终，我们希望本手册将帮助研究人员的斑马鱼组织操纵的细腻自然。

发育生物学研究人员利用的细胞，分子和遗传方法繁多揭开控制生物体是如何形成的机制。在这些方法中，组织操纵是对破译细胞命运，细胞运动和组织的组织复杂的问题的一个重要工具。以改变局部组织环境中的一个方法是通过用于输送蛋白质或其他信号分子1的焦点源微珠的外科应用。这种类型的实验操作中已被广泛实施的古典脊椎动物胚胎学模型，如青蛙和鸡2。 斑马鱼已经成为一个重要的脊椎动物模式生物器官的研究，并且还提供了许多独特的优势，为疾病建模3-5，因为它们共享高遗传保护与人类6。特别是，在光学透明性和移斑马鱼胚胎发育ternal提供了一个无与伦比的观点为观察组织个体发育3-5。大型向前遗传筛选的实施产生了斑马鱼的突变株用于进一步研究7,8和替代筛选技术鉴定，可在缩小的比例在单个实验室9,10高效率地进行了强有力的信息库。进一步的实验工作，斑马鱼已经通过在转基因方法的进步促进和反向遗传方法11,12，以及化学遗传学13-15。 组织操纵技术，如微珠的实施，还没有被作为广泛使用在斑马鱼，但仍然提供一个有用的工具，在开发过程中，以进一步了解细胞信号。微珠注入已用于询问器官形成的过程中的斑​​马鱼视网膜16,17，18心脏，脑19-22，神经嵴23和24,25鳍。在这些和其它研究中，珠已在开发过程中施加于理解信号分子26，梯度如何影响细胞迁移27和轴向图案28的扩散。最近，微珠已经用于评价再生机制在斑马鱼成年人29。在发展的研究，例如，斑马鱼微珠工作经历的胸鳍25研究提供了洞察肢体形成的机制。斑马鱼的胸鳍芽是同源的鼠标30和31小鸡前肢芽。脊椎动物肢芽有两个重要的信号节点：偏光活动（ZPA），通过声波刺猬表达建立前到后轴（SHH）和下游同源盒基因的目标区域，和顶外胚层脊（AER）的存在于肢芽，其作用通过的成纤维细胞生长因子的表达（的FGFs）建立邻近肢体的远端同一性的前端，通过植入FGF浸泡微珠到斑马鱼的Shh的遗传突变体，调查确定FGF作为必不可少的细胞周期的脊椎动物肢25和生长的进展。除了 FGF 和Shh信号级联位置设立标识，标识使用维甲酸（RA）小鸡肢芽作为分子，可以模仿偏振区域的作用，建立从前到后的身份32先驱性研究。这些实验涉及配售RA浸泡的Whatman纸小条放入鸡肢评估数字图案32。此外，研究人员已经进行使用使用微珠的其他优雅的研究，细胞移植，和外源性在斑马鱼的RA治疗，以确定的RA作用是斑马鱼后脑和中胚层28内提供远程位置线索。但是，目前仍有许多问题有关期间脊椎动物发育的许多方面类似的信号FGF和RA因素的作用。 RA的信令的影响，作为一个形态发生，影响许多器官33，如发育的心脏34和肾脏祖细胞，其中RA指定近端肾细胞类型的命运35-39。等主题进一步了解可以使用组织操纵和微珠植入技术实验研究显著受益。 而相比于像小鸡模型更少的研究已经完成与微珠植入在斑马鱼，那些已进行过高度信息。其中一个原因微珠植入根据调研，在斑马鱼胚胎的缺乏是likely表示有困难的技术挑战，根据胚胎的尺寸，从而构成一个障碍成功执行这样的操作的概念。然而，在斑马鱼胚胎微珠注入可以与实践而获知，并通过肉眼观察的技术中的辅助，因此可以追求作为询问发展的机制的装置。在这里，我们证明了精确的施用微珠进入斑马鱼胚胎，其可用于进行各种各样的组织形成和细胞形态测定的。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium Chloride</td>
			<td>American Bioanalytical</td>
			<td>AB01915</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>American Bioanalytical</td>
			<td>AB01652</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>American Bioanalytical</td>
			<td>AB00366</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Life Science</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4333-20ML</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Phenylthiourea (PTU)</td>
			<td>Aldrich Chemistry</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate (Tricane)</td>
			<td>Fluka Analytical</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass</td>
			<td>Sutter Instruments Co.</td>
			<td>BF100-50-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Flaming/Brown Micropipette puller</td>
			<td>Sutter Instruments Co.</td>
			<td>Mo. P097</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>Invitrogen</td>
			<td>15510-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene Blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140</td>
			<td></td>
		</tr>
		<tr>
			<td>250mL Erlenmeyer Flask</td>
			<td>Fischer Scientific</td>
			<td>FB-500-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Diposable Petri Dishes, Sterile</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>60mm x 15mm</td>
			<td>VWR</td>
			<td>25373-085</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm x 15mm</td>
			<td>VWR</td>
			<td>25373-100</td>
			<td></td>
		</tr>
		<tr>
			<td>150mm x 15mm</td>
			<td>VWR</td>
			<td>25373-187</td>
			<td></td>
		</tr>
		<tr>
			<td>Saint-Gobain Chemware Microspatula</td>
			<td>Fischer Scientific</td>
			<td>21-401-50B</td>
			<td></td>
		</tr>
		<tr>
			<td>P-1000 Micropipette tips</td>
			<td>Fischer Scientific</td>
			<td>2707402</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Temperature Incubator</td>
			<td>Fischer Scientific</td>
			<td>11 690 516DQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethly Sulfoxide</td>
			<td>American Bioanalytical</td>
			<td>AB00435</td>
			<td></td>
		</tr>
		<tr>
			<td>Microbeads (45-106 &#181;m)</td>
			<td>Biorad</td>
			<td>140-1454 AG1-X8</td>
			<td></td>
		</tr>
		<tr>
			<td>Microbeads (45 &#181;m)</td>
			<td>Polysciences</td>
			<td>7314</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Tweezer</td>
			<td>Roboz Surgical Instruments Co.</td>
			<td>RS-5010</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten Needle</td>
			<td>Roboz Surgical Instruments Co.</td>
			<td>RS-6065</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary tube holder</td>
			<td>Globe Scientific Inc.&#160;</td>
			<td>51674</td>
			<td></td>
		</tr>
	</tbody>
</table>

microbead implantation in the zebrafish embryo

斑马鱼已成为发育生物学和疾病的研究提供宝贵的遗传模型系统。斑马鱼共享高度的基因组保护，以及相似的细胞，分子和生理过程，与其他脊椎动物（包括人）。在个体发育早期，斑马鱼的胚胎是光学透明的，使研究人员能够想象器官的使用简单的实体显微镜的动态。微珠注入是一个方法，使组织操纵通过的因素在本地环境的改变。这使研究人员能够测定的任何数目的感兴趣的信号分子，如分泌肽，在发育中的胚胎内的特定空间和时间点的影响。在这里，我们详细的协议如何在早期斑马鱼发育操纵和植入珠。

几种类型的操作工具是用于在研究应用（ 图1）处理微珠有用。此外，一个简单的琼脂糖模具与小孔可以用来容纳斑马鱼胚胎，这是为了在一个及时和有效的方式（ 图1）上执行这些实验是至关重要的。微珠的植入可在空间的位置和感兴趣的时间阶段，这使得研究人员能够缩小动作的一个特定的窗口为感兴趣的分子来进行。 这里，单微珠注入到斑马鱼的胚胎在尾芽阶段或在期间体节形成阶段（ 图3）以后的时间点。两个不同尺寸的微珠，漂洗单独林格氏溶液，在该协议的示范（ 图3A，3B和3C）被使用。在没有任何共轭的本微珠注入小分子表明可以在没有形态学效应的总的斑马鱼胚胎（ 图4）的正常发育过程中可实现珠植入。 该实验主义者可能会遇到的困难与操纵微珠到由钨针之上的胚胎做了穿刺孔。为了达到更好的处理，一旦放置在胚胎由微针的微珠，晶须/睫毛工具都可以使用。这可以用自然脱落猫或犬晶须被制作，虽然其它天然或合成的间隙细丝可以被利用（ 图1）。不管是用钨针或晶须工具微珠的轻轻一推应该看到微珠沉入模具的林格氏液。一旦微珠​​下沉到模具它应该被移动大的距离，如果必要使用钨针，一旦上盖的胚胎针或晶须工具可以用于position为微珠成胚胎。微珠植入的成功可以立即通过可视化在立体显微镜来衡量，如珠将继续稳定地被定位在组织中。为了评价随时间珠位置，每个胚胎样品可通过实时的时间-过程摄影或成像以周期性的间隔检查（如在图4中示出），以确保珠位置没有偏移在实验的持续时间，由于内源性组织形态。理解珠随时间的位置是的结果，如评估小分子上的靶组织或发育过程中的作用的最明智的解释是至关重要的。 使用这些上述步骤将提供一个理想的环境，在其中嵌入微珠进入斑马鱼胚胎在期望的时间和位置。根据我们的经验，这种微珠植入协议的新手用户通常获得的技能NEcessary实践后，大约一个星期来执行微珠一致的手术植入到斑马鱼胚胎。 <img alt="图1" src="/files/ftp_upload/52943/52943fig1.jpg" /> 图1.工具用于执行微珠植入术。（一 ）珠植入托盘（左，直径15厘米）与胚胎模具（左）和微珠保温托盘（右侧，直径6厘米）。本工作托盘使研究者能够将胚胎成微珠植入所需的方向，并且具有准备微珠用于植入在显微镜台附近。微珠应孵育小分子（S）和/或车辆，然后将它们放置在被定位在显微镜台下胎圈注入托盘之前漂洗在单独微珠板（右）。 （B）的两对细镊子将用于去除蝶理在围绕每个斑马鱼胚胎和也打破微细针的末端。 （C）钨针会被用来做一个很细穿刺进入，在其中放置微珠的斑马鱼胚胎。 （ 四）微细移液管将用于拿起并在穿刺部位附近的斑马鱼胚胎的顶部位置微珠。 （ 五）晶须/睫毛工具将允许微珠到先前由钨针做胚胎内切口部位的温柔定位。 <a href="https://www.jove.com/files/ftp_upload/52943/52943fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52943/52943fig2.jpg" /> 图2.微珠植入过程中。（A）微珠植入托盘微珠soaki纳克在所需的洗涤液是建立一个立体显微镜下站，并定位与组织朝上的胚胎。 （B）使用一个钨针，小切口进入胚胎可以有轻微的但有力的运动进行。 （c）使用微毛细管移液管，所需的微珠可以拿起从微珠保温室中的托盘中，位于右侧，并提请左侧车厢，其中胚胎工作区位于。微珠应在切口部位附近​​沉积，然后轻轻地放置到切口部位使用晶须/睫毛工具钨针制成。一旦微珠被操纵进入切口部位，应当折入组织（远离切口），以稳定地定位的微珠和防止随后移动出作为发展继续的胚胎。 <a href="https://www.jove.com/files/ftp_upload/52943/52943fig2large.jpg" target="_blank">P</a>租赁点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/52943/52943fig3.jpg" /> 图3.微珠可植入胚胎在不同发育阶段。（A）的斑马鱼胚胎植入的微珠（大约50微米直径）到主干在尾芽阶段。当24小时后珠植入（hpbi）检查，胚胎已经发育正常和微珠的发育时间的推移过程中表现出最小的运动。在3体节阶段（β）植入微珠（直径大约50微米）到卵黄囊（B）的斑马鱼胚胎显示具有相对较小的珠运动正常发展随时间。 （C）的放大微珠（大约70微米直径）注入到7 SS胚胎同样显示出正常后续的发展，以及迷你如果MAL从植入网站的任何动静。 <a href="https://www.jove.com/files/ftp_upload/52943/52943fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52943/52943fig4.jpg" /> 斑马鱼胚胎的图4.总的发展是无扰由微珠通过72 hpbi存在下进行。斑马鱼的胚胎植入浸泡在林格氏溶液（直径大约50微米）微珠在7体节阶段（β）。每个微珠是手术插入树干，体节3和4之间的珠的存在与未在24小时后珠植入物（hpbi）公开发育缺陷，48 hpbi，或72 hpbi关联。 <a href="https://www.jove.com/files/ftp_upload/52943/52943fig4large.jpg" target="_blank">请点击此处查看更大的版本这个数字。</a>

Watch this Scientific Journal Video about Microbead Implantation in the Zebrafish Embryo at JoVE.com

Microbead Implantation in the Zebrafish Embryo

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

微珠植入斑马鱼胚胎

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

microbead-implantation-in-the-zebrafish-embryo

Research

JoVE Journal

Developmental Biology

11.1K 次观看.  University of Notre Dame. The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

视频: Microbead Implantation in the Zebrafish Embryo

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 ...

科研

发育生物学

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

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

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

观察有丝分裂和动力学在现场斑马鱼胚胎

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

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

胚胎显微注射和电的脊索动物<em&gt;玻璃海鞘</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.

Immunolabeling methods to analyze distinct populations of microtubules in the developing zebrafish brain are described here, which are broadly applicable to other tissues. The first protocol outlines an optimized method for immunolabeling stable and dynamic microtubules. The second protocol provides a method to image and quantify nascent microtubules specifically.

利用 Immunolabeling 分析斑马鱼胚胎中的稳定、动态和初生的微管

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

斑马鱼胚胎卵黄细胞中的 AFM 和 Microrheology

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario)

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on fish abdomens to expel the eggs. An alternative method uses the pressure of gas injected into the body cavity, which causes the subsequent release of the eggs. This method is believed to have less negative effects on both the welfare and egg quality of the broodstocks. Herein, we compare the results of air and hand stripping methods with respect to one-year survival and egg quantity and quality in two salmonid fish, rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta morpha fario). Our results indicate that air stripping yielded a better quality of eggs and higher one-year survival rate in rainbow trout. In addition, air stripping resulted in lower mortality rate than the group subjected to hand stripping (25% vs. 35%). The pH and hatching rate of the hand stripped group was lower than those of the air stripped group. In the case of brown trout, the quality of eggs obtained by both hand and air-stripping methods was similar; however, the one-year losses in fish were higher in air stripped group (15% compared to 0% in hand stripped fish). Although the advantages of air stripping method over hand stripping in terms of egg quality might not be observed in all salmonid species, the air-stripping procedure might be a promising option to be adopted in hatcheries as it ensures a high level of reproducibility and efficiency.

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout Oncorhynchus Mykiss and Brown Trout Salmo Trutta Morpha fario

The principal aim of this study is to standardize and test the pneumatic method (air stripping) of collecting eggs in rainbow trout and brown trout. This method allows effective and simple collection of the eggs without the necessity of fish abdomen massage.

两鲑鱼鱼、虹鳟 (虹鳟鱼虹鳟鱼) 和褐鳟 (鲑鲑形态石斑鱼) 的空气剥离效果

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...