Name: dual labeling of neural crest cells and blood vessels within chicken embryos using chickgfp neural tube grafting and carbocyanine dye dii injection
Uploaded: 2015-05-28T13:00:00.000+00:00
Duration: 9 min 57 s
Description: Watch this Scientific Journal Video about Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection at JoVE.co

受精的GFP鸡蛋由英国罗斯林研究所和爱丁堡大学的海伦·桑教授提供。罗斯林转基因鸡工厂由威康信托基金和生物技术和生物科学研究理事会（BBSRC）资助。这项工作的部分资金，和NT支持，由大奥蒙德街医院儿童慈善，伦敦，英国。作者感谢UCL儿童健康研究所的本·杰万斯帮助准备胚胎移植。

1. 神经管消融的微刀制备
<ol><li>用市售的钢缝纫针塑造微型刀具。<ol>
<li>首先使用安装在电动台式磨床上的研磨轮将针头两侧夷为平地。</li>
		<li>开始塑造手术刀，首先在粗级阿肯色州石头上使用控制的圆形运动，在针的两侧，在交替的方向。</li>
		<li>在额外的精细级阿肯色州石头上进行相同的锐化运动，以塑造一个超精细的微型刀片，具有明确定义的尖端（图1A，B）。 注:微型刀片的替代品可以是电镀金针、市售钨针或拉玻璃针。</li>
	</ol>
</li>
</ol>2. 将野生类型和 GFP 鸡蛋孵化到所需的阶段
<ol><li>在孵化前，在冷却的孵化器中储存受精鸡蛋和转基因 GFP 鸡蛋，因为在此温度下发育已停止。储存鸡蛋几天，最多一周。</li>
	<li>要开始开发，将野生类型和 GFP 鸡蛋水平放置在托盘上，同时在潮湿的（58 - 60%） 中孵化孵化器在37.5°C，使胚胎处于匹配阶段的神经管移植。</li>
	<li>为了在10-12个卵子发育阶段获得胚胎进行阴道神经管移植，孵卵1.5天（33-38小时），并根据汉堡和汉密尔顿21的发展表阶段胚胎。</li>
</ol>3. 准备鸡蛋用于窗口和移植
<ol><li>一次移动一个鸡蛋到定制鸡蛋架上进行窗口。用直剪刀反复敲击蛋壳上的一个小孔，在蛋尖端的上表面。</li>
	<li>用181~2克皮下针和5毫升注射器从鸡蛋中取出2-3毫升白蛋白。去除白蛋白可降低卵子内的蛋黄，并有助于随后的窗口，而不会对胚胎造成任何损害。<ol>
<li>丢弃白蛋白。用一小条透明胶带密封孔，用细剪刀切成大小。</li>
	</ol>
</li>
	<li>使用弯曲的剪刀，在蛋壳的上表面再挖一个洞。将剪刀尖插入孔中，使剪刀与长凳平行，以圆形运动将直径约 2 厘米的窗户切入外壳顶部。<ol>
<li>将剪刀保持静止位置，旋转鸡蛋。丢弃被移除的蛋壳盘。在E1.5，胚胎可以识别为蛋黄顶部的深黄色圆盘。</li>
		<li>使用钳子清除掉在鸡蛋内的任何贝壳碎片。丢弃任何未受精的卵子（由浅黄色蛋黄顶部的一个小白点识别）。</li>
	</ol>
</li>
</ol>4. 准备主机胚胎接受移植组织
<ol><li>将立体显微镜调整到眼睛水平，优化牙周光源的方向，在不引起反射的情况下充分照亮胚胎。</li>
	<li>为了使胚胎 正常可视化，使用口管和拉玻璃微管（图1C，Oii）在较深的黄色圆盘中心下注入少量印度墨水。<ol>
<li>准备墨水 50:50 与 PBS 含有青霉素/链霉素在 100 μg/ml 最终浓度。将微移液通过蛋黄膜插入胚胎外围，然后小心地将其尖端直接角在胚胎下面。</li>
		<li>通过吹在口腔管上，在胚胎下面输送墨水。如果不允许口腔管管，请改为使用 1 毫升注射器。小心不要在胚胎下面引入任何可能导致污染的气泡，然后小心地取出玻璃微管。这是一个微妙的步骤，如果不精确完成，可能导致胚胎死亡。</li>
	</ol>
</li>
	<li>参照汉堡和汉密尔顿21 来分阶段胚胎，并在实验室书籍中记录该阶段。</li>
	<li>使用安装在针架上的定制微型刀片（或细钨针），在进行微手术的区域旁边的维特尔线膜中制作一个非常小的气喘。<ol>
<li>小心地将2 -3滴PBS涂在膜撕裂上（使用玻璃微移液器和口腔管），在胚胎和膜之间创造空间。在膜中切下一个更大的窗口，以暴露将进行微手术的整个区域。</li>
	</ol>
</li>
	<li>使用微型脚轮去除感兴趣的神经管区域，从整个后部神经管的横穿孔和剖面切口开始（在视频中为 somite 1 到 7 的水平）。<ol>
<li>在神经管和索米之间进行双边切割，将神经管与周围组织分离，而不会损坏索米特。</li>
		<li>非常轻轻地将神经管与底层的神经管分开，后者应保持完好无损。请注意，成功的神经管切除将保持所有周围的组织完好无损（图2）。</li>
		<li>将切除的神经管吸进玻璃微管中，然后丢弃。</li>
	</ol>
</li>
	<li>在实验室书中记录神经管消融水平。宿主胚胎现在准备接受供体神经管。</li>
</ol>5. 准备捐赠移植组织
<ol><li>通过使用 FITC 滤镜在荧光立体显微镜下观看，选择经过窗口的舞台匹配的 GFP 胚胎。GFP荧光使得它很容易想象烟体和阶段的胚胎。</li>
	<li>一旦确定了一个相匹配的胚胎，通过做4个切口从卵子中取出胚胎，用帕舍夫-沃尔夫弹簧剪刀（图1C，l）在胚胎周围的矩形形状，然后用胚胎勺轻轻拾起它。</li>
	<li>将胚胎放在带硅藻聚合物底座的方形表玻璃中。用杜蒙#5钳子轻轻摇动胚胎，去除任何附着的蛋黄。取出维他林膜，用不锈钢细针（图1C）将胚胎固定在聚合物底座上。</li>
	<li>使用弹簧剪刀，在神经管和周围索米特周围以矩形形状切开4个切口，位于从宿主胚胎中取出的相同区域。</li>
	<li>使用塑料转移移液器，将神经管和松虫组织从供体GFP胚胎转移到含有0.2%胰腺素的手表玻璃中。</li>
	<li>允许酶消化在RT进行10分钟，以帮助分离组织。在酶中孵育后，使用安装在手柄上的不锈钢小针手动将神经管与所有相邻组织分离。</li>
	<li>使用玻璃微管，将分离的神经管转移到另一个手表玻璃中，其中含有10%的血清（如山羊、马或胎儿小腿），以冲洗多余的胰腺素并停止酶消化。5分钟后，解剖的神经管准备移植到小鸡主机（图2和S1）。</li>
</ol>6. 移植组织
<ol><li>使用玻璃微管，小心地将解剖的神经管从表玻璃转移到主机胚胎。将神经管置于正确的前后方向，并使用微型刀块轻轻推送与小鸡宿主切除区域相邻的外植。将一小块等体附着在后部表面，或切入一个小刻痕，以识别神经管的方向。<ol>
<li>如有必要，使用微型刀刀将去除的植物修剪到被切除区域的确切大小。</li>
	</ol>
</li>
	<li>轻轻地引导神经管进入消融区域，并定位它，使后侧正确定向。使用安装在口腔管上的玻璃微移液器去除移植物周围的 PBS 和/或液体。这有助于捐赠者和宿主组织坚持和移植成为建立。</li>
	<li>用24毫米宽的透明胶带密封整个窗户，以防止脱水和污染。</li>
	<li>用铅笔在蛋壳上标记，并在实验室书上记录其数量，从而给幻想胚胎贴上标签。将鸡蛋返回孵化器以进一步发展。</li>
</ol>7. 将DiI注射到宿主胚胎的血管中
<ol><li>在所需的实验时间点（这里，3 - 10天后），从孵化器中取出幻想胚胎，然后用直剪刀取出透明胶带，以进入卵子内的胚胎。</li>
	<li>如有必要，使用剪刀放大外壳中的窗口。小心不要损坏胆囊膜，如果它连接到壳，这将导致出血和危及血管标签。</li>
	<li>在蛋黄上选择一个可访问的静脉，确保血液流向胚胎。选择其中一个脉脉的分支点（图3B，C）。 注:在 E6.5 - E7.5 时，可能需要用钳子轻轻将胆囊膜移到一边才能进入蛋黄静脉。E8.5之后，唯一的选择是注射到胆囊膜静脉之一，因为，在这个阶段，胆囊膜完全覆盖胚胎。</li>
	<li>使用两个杜蒙#5钳子在选定的注射点上方取出维特尔线膜，向相反的方向撕裂。</li>
	<li>使用 Dumont #5打破拉玻璃针，并在加载 CellTracker CM-DiI 之前将其直径调整到静脉的大致大小。在 DMSO 中以 40μg/μl 的速度制作 DiI 库存解决方案，并存储在 -20 °C。 以浓度为 4μg/μl 的 0.3 M 蔗糖/PBS 准备工作解决方案。<ol>
<li>使用口管吸吸，在 0.3 M 蔗糖/PBS 中吸入 5 - 10 μl 的 DiI。较老的胚胎可能需要高达25微升或更多。从E8.5开始，胚胎有更大、更肌肉的静脉，可能需要用杜蒙#5保持位置，然后用装有DiI的玻璃针刺住。</li>
	</ol>
</li>
	<li>迅速将针头插入静脉，用口管稳定地吹，使 DiI 能够缓慢地加入血液流动，而不会形成血块。或者，使用压力喷油器进行 DiI 交付。</li>
</ol>8. 分段或全山检查的收获胚胎
<ol><li>为了在胚胎内保留尽可能多的DiI，注射后立即收获胚胎，将其铲入穿孔的勺子上，用一把直剪刀切割血管和结缔组织，将胚胎从蛋黄中解放出来。</li>
	<li>取出任何松散的膜，解剖出感兴趣的器官（即本教程中的肺和消化道），非常小心不要压缩组织，这会产生DiI的扩散。立即修复组织浸入4%PFA 1-2小时在RT。</li>
	<li>在 PBS 中冲洗组织 5 分钟，然后在含有 5μg/ml DAPI 的 PBS 中冲洗 15 分钟。将样品安装在桥接显微镜滑梯上进行整个安装检查或嵌入样本进行低温剖面。</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytodifferentiation+of+quail+tectal+primordium+transplanted+homotopically+into+the+chick+embryo.">Cytodifferentiation of quail tectal primordium transplanted homotopically into the chick embryo.</a> Brain Research. 429, 187-205 (1987).</li><li>Wu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inter-species+grafting+caused+extensive+and+heritable+alterations+of+DNA+methylation+in+Solanaceae+plants.">Inter-species grafting caused extensive and heritable alterations of DNA methylation in Solanaceae plants.</a> PLoS One. 8, e61995 (2013).</li><li>Freem, L. J., Delalande, J. M., Campbell, A. M., Thapar, N., Burns, A. 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作者宣称他们没有相互竞争的经济利益。

物种内部神经管移植方法，结合此处描述的血管标记，充分利用卵子内鸟类胚胎（与其他脊椎动物胚胎相比）的易于获取，研究自主神经系统（ENS）和血管系统元素的共同开发。
对于NCC衍生物的标记，我们描述的小鸡GFP-鸡种内嫁接方法比40多年前1-3年建立的经典鹅鸡奇美拉方法具有许多优点。首先，在 FITC 光下，GFP 荧光非常明亮，以至于 GFP® 细胞在活体幻想胚胎中很容易识别。这使得移植的成功在奥沃检查，而鹅鸡移植需要使用QCPN杀死，加工和免疫，然后才能确定移植的成功2。其次，转基因小鸡GFP中的GFP表达是细胞质的，因此它不仅给细胞体贴上标签，而且使移植细胞的投影可视化22。这允许以高分辨率观察复杂的神经元网络（请注意，当样品中沾有抗GFP抗体时，精细投影最好可视化）。由于 QCPN 标签仅限于夸尔细胞核，因此不会使用夸尔-奇美拉显示此类网络。第三，物种内部移植消除了奇美胚胎细胞之间的任何潜在物种差异。由于小鹰胚胎的潜伏期比小鸡短（19天对21天），因此有人建议，小鹰细胞的增殖率高于小鸡细胞，这可能影响到23个奇美组织的发展。有趣的是，在植物中也表明，物种间嫁接可以产生广泛的DNA甲基化模式在宿主24的变化。第四，小鸡GFP促进背部移植实验，以解决诸如NCC命运和细胞承诺25等话题。第五，转基因小鸡GFP对许多其他技术也很有用，包括GFP®细胞亚群的FACS排序、含有GFP®细胞的器官的器官培养、通过表达质粒26的电波对GFP®移植组织的基因操作，以及其他成像技术，如光学投影断层扫描27。
神经管移植方法可以通过微手术取代较短的神经管来修改。通过使用较小的神经管段，显微外科对胚胎的损害可能较小，存活率可以提高。然而，移植较少神经管的缺点是，在宿主中GFP+NCC的数量将减少。用户可以尝试在移植神经管以提供胚胎最佳存活率的神经管数量和主肠道内 GFP® NCC 的数量之间取得平衡，以提供翔实的结果。
对于血管绘画，DiI 的优势在于其荧光非常明亮和坚固。此外，它有能力在固定期间扩散，确保最好的开放毛细细丝染色。由于胚胎是一种重要的染料，因此可以在注射过程中存活下来，并使用染色血管系统继续发育（我们手中长达24小时，虽然随着时间的推移，染色变得更加刺穿，见 图3E）。因此，小鸡GFP 嫁接与DiI血管绘画的结合与活影像相容。除了所有这些优点，重要的是要注意，血管注射只标记发光血管，因此不识别未开封的毛细血管，内皮尖端细胞或孤立的内皮细胞。然而，在鸟类转基因的进一步进展可以提供新的方法来规避这些问题，例如实验使用Tg（tie1:H2B-eYFP）的鹅胚胎来研究血管形态形成28。这项技术的另一个局限性是，对于E7.5及以后胚胎的有效血管标签，需要注射更多的染料，这会使实验变得昂贵。然而，这项技术的修改可能包括低成本的血管标签使用荧光笔墨水14，虽然这种方法还没有尝试在我们的手中。
程序的关键步骤包括通过在爆炸盘下注入墨水来可视化胚胎的过程。如果覆盖蛋黄的膜在这个阶段被充满墨水的针头撕裂，那么胚胎存活率就会受到严重损害。此外，在准备供体神经管时，组织不会在胰腺素中留下过长的时间（考虑最多大约 10 分钟）。长期暴露在胰腺素损害组织和神经管然后很难处理，它不会很好地融入主机。在注射奇美胚胎之前，获得野生类型胚胎的DiI注射技术经验至关重要，因为每个胚胎通常只能尝试注射一次。DiI体积和针直径是每个胚胎的关键参数，应在野生类型、阶段匹配控制上进行评估。
总之，我们在活小鸡胚胎中采用神经管移植和DiI血管绘图的双重标记方法，可用于研究器官形成过程中NCC与血管网络之间的相互关系。考虑到在器官发育过程中建立正确目标内向和血管化的机制仍鲜为人知，这种方法为该领域的未来发现具有潜力。

鸡胚胎是脊椎动物发育生物学中一种宝贵的模型生物体，尤其是因为它 在卵母体内 的发育允许实验操作，否则在 子宫内发育的脊椎动物中是不可能执行的。这种可访问性和易于操作性导致小鸡胚胎在发育生物学领域的许多开创性发现中扮演关键角色。其中最强大的技术是利用小鸡的奇美胚胎来研究细胞命运，这是妮可·勒杜阿林教授在20世纪70年代的1-3年开创的方法。特别是，在早期发育期间，小鸡奇美拉在基因标记和跟随高度迁移神经峰细胞（NCC）种群方面特别有用。NCC是一种多能的迁移细胞群，生长在神经管边缘的后部细胞群中，在整个脊椎动物胚胎中产生多种细胞类型。这些结构包括颅面结构（软骨、骨骼、肌肉）、神经元和胶质（在感官和自主神经系统中）、黑色素细胞，以及内分泌系统2，4，5细胞的亚群。影响NCC命运的最重要因素之一是它们沿着神经管前后轴的初始位置。例如，产生肠神经系统（ENS）神经元和胶质的肠道NCC来自两个离散的亚种群:第一个位于虚无（caudal后脑）区域，第二个位于神经管6-13的囊性区域。神经管相应区域的物种间嫁接是永久标记这些细胞并随后允许跟踪这些细胞的技术，从它们诞生于神经管的边缘，到消化道6，7，10内的最终目的地。
与其他动物模型相比，另一种胚胎操作更容易在小鸡体内进行，是血管系统的重要标签。事实上，随着小鸡胚胎的发育，它位于一个胚胎外血管网络之上，该血管网络从蛋黄中循环氧气和营养物质。这个无障碍血管网络，位于蛋黄表面，可以用作在器官发生12，14-17期间标记胚胎发育血管系统的网关。各种染料（如嗜脂染料 DiI）的血管内注射，使描述/染色新生血管网络的所有发光血管成为可能。
由于发育器官需要连接到神经系统（用于感觉和运动控制）以及血管系统（用于气体交换、液体和营养供应），因此两个网络相互发展，并在其分支结构18-20中具有惊人的相似性。在这里，我们报告胚胎操作，使我们能够研究同时开发NCC衍生的ENS，以及血管系统，在器官形成期间。这是通过移植神经管的离散部分（包括神经峰）以及血管DiI注射来产生鸡幻想。作为从鹅鸡奇美拉的进展，我们的方法使用转基因GFP小鸡胚胎进行物种内部移植，使移植技术在成像细胞及其投影方面更加强大，并消除与物种差异相关的任何潜在偏差。

<table><tbody><tr><td>Fertilised chick eggs</td><td>Henry Stewart and Co, Louth, UK</td><td></td><td></td></tr><tr><td>Fertilised GFP chick eggs</td><td>The Transgenic Chicken Facility, The Roslin Institute, The University of Edinburgh</td><td></td><td></td></tr><tr><td>Egg incubator (Profi-H Hatcher)</td><td>Lyon Technologies, CA, USA</td><td>910-033</td><td></td></tr><tr><td>14C Incubator</td><td>Precision Cooled Incubator, Leec Ltd., Nottingham, UK</td><td>Model LT2</td><td></td></tr><tr><td>Stereo-microscope&amp;nbsp;</td><td>LEICA</td><td>Model MZ 12.5</td><td></td></tr><tr><td>Digital Camera</td><td>LEICA</td><td>DC500</td><td></td></tr><tr><td>Image acquisition software</td><td>LEICA</td><td>IM50</td><td></td></tr><tr><td>Goose neck halogen cold light source</td><td>Advanced Imaging Concepts, Inc</td><td>KL 1500 LCD&amp;nbsp;</td><td></td></tr><tr><td>181&amp;frasl;2 G hypodermic needle&amp;nbsp;</td><td>SIGMA - ALDRICH</td><td>HSWNH181</td><td></td></tr><tr><td>Pancreatin</td><td>SIGMA - ALDRICH</td><td>P3292</td><td></td></tr><tr><td>DMEM</td><td>SIGMA - ALDRICH</td><td>D5030</td><td></td></tr><tr><td>Goat serum</td><td>SIGMA - ALDRICH</td><td>G6767</td><td></td></tr><tr><td>5 ml syringe</td><td>SIGMA - ALDRICH</td><td>Z248010</td><td></td></tr><tr><td>Mouth tube</td><td>SIGMA - ALDRICH</td><td>A5177</td><td></td></tr><tr><td>Sigma Pasteur pipettes non-plugged, L 5 3/4 in.</td><td>SIGMA - ALDRICH</td><td>S6018</td><td></td></tr><tr><td>Transfer pipettes, polyethylene</td><td>SIGMA - ALDRICH</td><td>Z350796&amp;nbsp;</td><td></td></tr><tr><td>Borosillicate glass capillaries, thin wall without filament</td><td>Harvard apparatus</td><td>PY8 30-0035</td><td></td></tr><tr><td>Iris Scissors - ToughCut</td><td>Fine Science Tools</td><td>14058-09</td><td></td></tr><tr><td>Curved Iris Scissors - ToughCut</td><td>Fine Science Tools</td><td>14059-09</td><td></td></tr><tr><td>Needle holders (Nickel-plated pin holder)&amp;nbsp;</td><td>Fine Science Tools</td><td>26018-17&amp;nbsp;</td><td></td></tr><tr><td>Pascheff-Wolff Spring Scissors&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;</td><td>15371-92</td><td></td></tr><tr><td>Dumont #5 forceps&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;</td><td>11251-30&amp;nbsp;</td><td></td></tr><tr><td>Minutien pins&amp;nbsp;</td><td>Fine Science Tools</td><td>26002-15</td><td></td></tr><tr><td>Dumont AA forceps, Inox Epoxy- coated&amp;nbsp;</td><td>Fine Science Tools</td><td>11210-10&amp;nbsp;</td><td></td></tr><tr><td>Perforated spoon</td><td>Fine Science Tools</td><td>10370-18</td><td></td></tr><tr><td>Tungsten needles (0.125mm diameter)</td><td>Fine Science Tools</td><td>10130-05</td><td></td></tr><tr><td>Sellotape (clear, 24 mm width)</td><td>Any Supplier</td><td></td><td></td></tr><tr><td>Pen/Strep (Penicillin, Streptomycin) Solution</td><td>VWR international</td><td>101447-068&amp;nbsp;</td><td></td></tr><tr><td>Sylgard 184 silicone elastomer kit</td><td>Dow Corning</td><td>S09 512 516</td><td></td></tr><tr><td>Pelikan black ink</td><td>Pelikan</td><td>211-169</td><td></td></tr><tr><td>CellTracker CM-DiI&amp;nbsp;</td><td>Molecular Probes</td><td>C-7001</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Molecular Probes</td><td>D1306</td><td></td></tr><tr><td>Settings for glass needle puller&amp;nbsp;</td><td>Sutter Instruments</td><td>Flaming/Brown micropipette puller model P-86</td><td></td></tr><tr><td>Heat 950; Pull 150; Velocity 100; Time 200; Pressure 500</td><td></td><td></td><td></td></tr></tbody></table>

dual labeling of neural crest cells and blood vessels within chicken embryos using chickgfp neural tube grafting and carbocyanine dye dii injection

所有发育中的器官都需要连接到神经系统（用于感觉和运动控制）以及血管系统（用于气体交换、液体和营养供应）。因此，神经系统和血管系统相互发展，并在其分支结构中有着惊人的相似性。在这里，我们报告胚胎操作，使我们能够研究神经波峰衍生神经组织（在这种情况下，肠神经系统）和血管系统的同时发展。这是 通过 移植神经管的离散部分和相关的神经峰，结合血管DII注射在同一胚胎实现的。我们的方法使用转基因小鸡GFP 胚胎进行物种内部移植，使移植技术比自20世纪70年代以来使用的经典小鸡间移植协议更加强大。小鸡GFP- 鸡种内嫁接有助于移植细胞的成像及其在完整组织中的投影，并消除与物种差异相关的细胞发育中的任何潜在偏差。该方法充分利用鸟类胚胎（与其他脊椎动物胚胎相比）的易于获取，研究肠道神经系统和血管系统的共同发展。

图1 显示了进行神经管显微外科隔离和移植所需的典型仪器。 图2 显示移植过程。移植胚胎移植后，将进行筛查，以获得移植成功。这涉及到在立体荧光显微镜下检查胚胎，通常是在显微外科后的第二天早上，是否存在移植衍生的 NCC。如果移植成功，那么GFP®NCC可以在神经管附近和通往前体的早期迁移路径中观察到。如果手术没有成功，GFP®NCC将不会在神经管外观察到，或者如果它们存在于主机中，它们的数量可能较小。这些不成功的胚胎被丢弃。通常，5-8个神经管移植在一天内进行，其中80%是成功的。神经管移植失败的原因包括胚胎在显微外科期间因组织损伤而死亡，或神经管无法融入宿主胚胎。后者可能是由于神经管在宿主体内的放置不良或由于解剖技术差或过度接触分离酶而导致的神经管质量差。最初的筛选步骤，以及GFP®细胞的类似后期检查，是有用的，因为它意味着时间和资源不会浪费通过对没有GFP标签的NCC在肠道内的胚胎进行实验。
图3显示血管DII注射的程序。DiI 注射技术的效率/成功取决于:首先，将注射针切到目标静脉的最佳直径，第二，在静脉中插入针头时做出精确的手势（以免穿透另一侧），第三，通过以恒定速度吹制针头，避免针头在注射过程中被堵塞。如果这三个参数中的任何一个做错了，胚胎将出血或需要几个小时才能恢复，然后再进行第二次尝试，因为出血使得几乎不可能立即重新注射。成功的胚胎应立即通过立体荧光显微镜下观察来选择，并且必须快速解剖。在成功的胚胎中，在整个胚胎（图3C，D）中都存在DiI标记的血管，包括毛细血管（图3D）。
在采集胚胎和检查组织部分或全山胃肠道后， 典型结果表明，GFP®NCC在原始ENS内，以及DiI标记肠道血管网络的精细结构（图4）全山制剂，可使用共聚焦显微镜进行检查，图像堆栈产生三维（3D）重建，显示GFP+ENS细胞精细投影与DiI染色血管系统（图4 A-C） 之间的相互关系:G-I;视频1和2）。
<img alt="Figure 1" src="/files/ftp_upload/52514/52514fig1.jpg"> 图1。推荐的显微外科仪器。 （A） 缝纫针形状的微型刀具。 （B） 精细阿肯色州石头塑造微型刀片。 （C） a） 直剪刀， b） 弯曲剪刀， c） 5 毫升注射器与 181×2 G 皮下针， d） 塑料移液器， e） 定制鸡蛋支架， f） 黑色墨水， g） 方形表杯， h） 方形手表玻璃与黑色银质基础， i） 微型刀片在针架上， j） 米努蒂安针， k） 迷你或钨针在针架上， l） 帕舍夫沃尔夫弹簧剪刀， m） 杜蒙#5钳子， n） 穿孔勺子， oi） 短火拉转移针， oii） 长火拉墨针， p） 口管。 <a href="https://www.jove.com/files/ftp_upload/52514/52514fig1large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" src="/files/ftp_upload/52514/52514fig2.jpg"> 图2。物种内部神经管移植。 小鸡胚胎/GFP神经管图像已经从德拉兰德 等人修改。12. 血管化是肠道神经峰细胞殖民化所必需的。 <a href="https://www.jove.com/files/ftp_upload/52514/52514fig2large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" src="/files/ftp_upload/52514/52514fig3.jpg"> 图3。静脉注射。（A）推荐仪器:a） 细胞跟踪器 CM-DiI 滴在胶片上，b） 拉玻璃注射针，c） 口管。（B）静脉注射到E4奇美小鸡胚胎的切图图。 （C） 在 ovo DiI 静脉注射中显示含有 DiI 插入静脉（箭头）的精细玻璃针。 （D） E4 芯片胚胎后 DiI 注射 （红色） 与 GFP® 神经管 （箭头）.（E） DiI在活胚胎中染色细血管网络，注射后24小时。布尔:大脑:H:心脏:LB:肢体芽:答:阿兰托瓦。 （C） 和 （D） 中的图像已从德拉兰德 等人处修改。12 血管化不是肠道殖民化的必要，由肠道神经波峰细胞。 <a href="https://www.jove.com/files/ftp_upload/52514/52514fig3large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" src="/files/ftp_upload/52514/52514fig4.jpg"> 图4:代表结果在E5.5小鸡胚胎的胃和腹。（A-C）胃部区域的共焦图像堆栈的三维 （3D） 重建显示 （D） GFP® 肠神经波峰细胞 （ENCC） （E） DiI 染色血管系统和（F） ）两个网络D-F组织学部分在胃的水平合并图像显示（G）GFP+ENCC（H）DiI染色血管系统和（I）两个网络的合并图像。 核上沾满了DAPI（青色）。（G-H）在 Caecum 区域对共焦图像堆栈进行 3D 重建，显示（A） GFP® ENCC 迁移前部为绿色，（B） DiI 彩色血管系统为红色，（C）两个网络的合并图像。图像（A-F）已经从德拉兰德等人修改。12血管化不是肠道殖民化的必要，由肠道神经波峰细胞。<a href="https://www.jove.com/files/ftp_upload/52514/52514fig4large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure S1" src="/files/ftp_upload/52514/52514figS1.jpg"> 图 S1。通过酶消化和微切除将供体GFP®神经管与周围组织隔离 。（A）从供体胚胎解剖的GFP®神经管和相邻的索米特。 （B） 胰腺素消化后隔离神经管，使用不锈钢小针进行微切除。所以:索米特斯:NT:神经管:诺托乔德。
<img alt="video 1" src="/files/ftp_upload/52514/52514video1.jpg"> 图 4C 中图像的 1. 3 维 360° 旋转视频，显示 E5.5 （HH27-28） 的血管系统和胃中的 ENCC。<a href="https://www.jove.com/files/ftp_upload/52514/JoVE_Video1.avi" target="_blank">请单击此处查看此视频。</a>
<img alt="Video 2" src="/files/ftp_upload/52514/52514video2.jpg"> 图4I中图像的三维360°旋转视频，显示E5.5（HH27-28）在Caecum区域的血管系统和ENCC迁移前。<a href="https://www.jove.com/files/ftp_upload/52514/JoVE_Video2.avi" target="_blank">请单击此处查看此视频。</a>

Watch this Scientific Journal Video about Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection at JoVE.co

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

在这里，我们报告神经波峰细胞和血管的双重标签使用小鸡GFP 神经管内部嫁接结合血管内DII注射。这种实验技术使我们能够同时在器官发生期间可视化和研究NCC衍生（肠）神经系统和血管系统的发展。

使用小鸡GFP 神经管移植和碳水化合物染料 DiI 注射，在鸡胚胎内对神经囊细胞和血管进行双重标记

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

dual-labeling-neural-crest-cells-blood-vessels-within-chicken-embryos

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

8.2K 次观看.  UCL Institute of Child Health. 在这里，我们报告神经波峰细胞和血管的双重标签使用小鸡GFP 神经管内部嫁接结合血管内DII注射。这种实验技术使我们能够同时在器官发生期间可视化和研究NCC衍生（肠）神经系统和血管系统的发展。 

视频: Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

跨物种移植神经嵴迁移和分化的分析

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

神经科学

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

在鸡胚神经诱导的含量为

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Creating Avian Forebrain Chimeras to Assess Facial Development

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the strengths of this model system is that the effect of, and interaction among, tissues can be directly assessed in chimeric embryos. We have previously shown that signals from the forebrain contribute to facial morphogenesis by regulating the shape of the expression domain of Sonic hedgehog (SHH) in the Frontonasal Ectodermal Zone (FEZ). In this article, the method of generating the forebrain chimeras and provide illustrations of the outcomes of these experiments is described.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of basal forebrain&#160;during craniofacial development.

创建鸟类前脑嵌合体以评估面部发育

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the ...

发育生物学

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

单细胞转染鸡胚

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

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

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

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

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

In Ovo Intravascular Injection in Chicken Embryos

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

In Ovo Intravascular Injection in Chicken Embryos

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

在 奥沃 鸡胚血管内注射

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

使用小鸡^GFP神经管移植和碳水化合物染料 DiI 注射，在鸡胚胎内对神经囊细胞和血管进行双重标记

摘要

探索更多视频

此视频中的章节

双整装原位杂交技术在早期鸡胚

跨物种移植神经嵴迁移和分化的分析

在鸡胚神经诱导的含量为

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

创建鸟类前脑嵌合体以评估面部发育

单细胞转染鸡胚

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

在奥沃鸡胚血管内注射

评估外胚层上皮细胞在颅面发育的信号属性

使用小鸡GFP 神经管移植和碳水化合物染料 DiI 注射，在鸡胚胎内对神经囊细胞和血管进行双重标记

摘要

探索更多视频

此视频中的章节

双整装原位杂交技术在早期鸡胚

跨物种移植神经嵴迁移和分化的分析

在鸡胚神经诱导的含量为

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

创建鸟类前脑嵌合体以评估面部发育

单细胞转染鸡胚

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

在 奥沃 鸡胚血管内注射

评估外胚层上皮细胞在颅面发育的信号属性

使用小鸡^GFP神经管移植和碳水化合物染料 DiI 注射，在鸡胚胎内对神经囊细胞和血管进行双重标记

在奥沃鸡胚血管内注射