Name: primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos
Uploaded: 2016-03-10T13:00:00.000+00:00
Duration: 11 min 53 s
Description: Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

资金目前的协议的发展特别是"目标为一流大学计划"国立台湾大学，台湾（授权ID-104R350144），以及台湾科学技术部（授权ID的支持:科技部104 -2313-B-002-039-MY3）。我们特别感谢中心国立台湾大学生物技术用于提供动物房和实验室空间，目前的研究。

注:此步骤是Bauer 等人 2013年和中泽等人 ，2011年2,9制定了鸡模型文化协议的修改。 1.准备健康胚胎第5天的胚胎从日本鹌鹑<ol><li>将1男3女性成熟的日本鹌鹑在同一个笼子里。供应饲料自由采食和饮水 。调整的光在动物室的光的14小时和黑暗10小时。 </li><li>每天收集在塑料袋受精卵在下午和保存在16℃的冰箱延缓胚胎的生长速度。 </li><li>两个星期的时间内收集足够数量的鸡蛋后，孵化受精卵在37℃培养箱具有良好的透气五天。 </li></ol> 2.准备基础培养基和冲洗溶液<ol><li>使用DMEM / F-12培养基（调节至pH 7.2）作为生长培养基，并补充它用10％新生小牛血清（NBCS）和1％青霉素 - 链霉素Ampho。溶液（PSA;青霉素，10 5单位/ L，链霉素，100毫克/升;两性霉素B，0.25毫克/升）。 </li><li>制备在10倍浓度为存储磷酸盐缓冲盐水（PBS）溶液;用1×PBS溶液（含NaCl，137.93毫;氯化钾，2.667毫; KH 2 PO 4，1.471毫; 的 Na 2 HPO 4 -7H 2 O，8.06毫摩尔;调整至pH 7.2）洗和滋润卵黄囊膜而分离蛋黄和白蛋白的内胚层。 </li><li>使用DMEM培养基（调节至pH 7.2）重新悬浮内胚层粒料。 </li></ol> 3.从日本鹌鹑YSMs主要EECS集<ol><li>孵育所收集的鸡蛋5天（步骤1.3）。检查由禽蛋蛋，确认胚胎正常发育。 注:在5天的胚胎，YSM中胚层的边界非常清晰，3胚胎层不太紧密联在一起，所以很容易OB泰恩内胚层。仅使用显示了YSM集合正常微血管循环发育的蛋。 </li><li>仔细清洁利用淡水和75％乙醇的蛋壳。打开由一对剪刀的空气电池的位置的蛋壳，轻轻剥离并通过在充满37℃1×PBS中在10cm培养皿镊子和地点收集整个YSM。 </li><li>用剪刀取出胚，蛋黄和蛋清，轻轻用1×PBS洗涤YSM三次，并放置YSM在10cm的培养皿PBS溶液。 </li><li>使用镊子和剪刀从YSM删除外胚层。使用2镊子，一个紧紧握住内胚层细胞层，而其他持有毛细管中胚层。 </li><li>在解剖显微镜下，拉开和内胚层从沿朝向胚胎的方向上的中胚层的边缘分开。收集用在15毫升生长培养基的一个滴管和地点在50ml离心管中的浅黄色内胚层在37℃笏呃洗澡，直到所有组织的集合完毕。 </li></ol> 4.内胚层片胶原酶消化消化<ol><li>新鲜溶解6.5单位IV型胶原酶的10ml DMEM中。 </li><li>离心在130 xg离心50毫升管（3.4），在室温3分钟。吸出中，用吸管把所有的内胚层在将6cm培养皿。加入1 ml胶原酶解决这个培养皿。 </li><li>切片来自2鹌鹑胚胎有一对弯曲的剪刀，直到每个片的大小收集的内胚层是大约2至3毫米。使用滴管，收集所有切片到50毫升离心管用9ml新鲜胶原酶溶液一起。 </li><li>消化胶原酶的组织30分钟在37℃振荡水浴中于175转。用胶原酶此部分消化用于提高外植体的细胞增殖。 </li><li>离心在130×g离心3分钟，在RT，轻轻用移液管除去上清液部分。通过悬浮于20毫升的DMEM洗涤沉淀并除去后130 XG离心上清液部分3分钟。 </li><li>重悬用12ml生长培养基（含10％NBCS和1％的PSA的DMEM / F12）的沉淀，轻轻地，同样接种到24孔板（每孔用500μl细胞溶液）。 </li><li>孵育组织外植体在37℃在5％CO 2的空气中2天，使细胞增殖出组织。 </li><li>孵育细胞另外2天，并通过细胞生存力测定法协议表征为细胞活力。15 注:活细胞保持细胞质内的还原环境。刃天青，活性成分，是无毒的，细胞透过性化合物是在蓝色和实质上非荧光。当加入到细胞培养物，刃天青是减少在由线粒体酶的胞质溶胶向试卤灵。试卤灵是红色和高荧光。活细胞不断转化为刃天青试卤灵，increASING周围细胞的培养基中的总荧光和颜色。 </li><li>执行内胚层上皮细胞总RNA分离，如所描述逆转录和实时定量标记基因mRNA表达的PCR。16,17 <ol><li>使用逆转录PCR检测SOAT1的mRNA（有义:5&#39;-GAAGGGGCCTATCTGGAACG-3&#39;;反义:5&#39;-ATCTGCACGTGACATGACCA-3&#39;; PCR产物= 168碱基对）作为欧洲经济共同体细胞的特异性标记物。使用β肌动蛋白的mRNA（有义:5&#39;-TGGTGAAGCTGTAGCCTCTC-3&#39;;反义:5&#39;-GTGATGGACTCTGGTGATGG-3&#39;; PCR产物= 151碱基对），为管家基因和内部对照。 </li><li>用凝胶电泳检测，RT-qPCR的的产品尺寸。制备的1％琼脂糖凝胶上，并用2微升样染料混合10微升的qPCR产物。负载10微升混合物倒入每个孔中的1％琼脂糖凝胶，并运行由100V的凝胶30分钟。 </li></ol></li></ol>

<ol>
	<li>Abeyrathne, E. D., Lee, H. Y., Ahn, D. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egg+white+proteins+and+their+potential+use+in+food+processing+or+as+nutraceutical+and+pharmaceutical+agents--a+review.">Egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical agents--a review.</a> Poult Sci. 92 (12), 3292-3299 (2013).</li><li>Bauer, R., Plieschnig, J. A., Finkes, T., Riegler, B., Hermann, M., Schneider, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developing+chicken+yolk+sac+acquires+nutrient+transport+competence+by+an+orchestrated+differentiation+process+of+its+endodermal+epithelial+cells.">The developing chicken yolk sac acquires nutrient transport competence by an orchestrated differentiation process of its endodermal epithelial cells.</a> J Biol Chem. 288 (2), 1088-1098 (2013).</li><li>Ding, S. T., Nestor, K. E., Lilburn, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+concentration+of+different+lipid+classes+during+late+embryonic+development+in+a+randombred+turkey+population+and+a+subline+selected+for+increased+body+weight+at+sixteen+weeks+of+age.">The concentration of different lipid classes during late embryonic development in a randombred turkey population and a subline selected for increased body weight at sixteen weeks of age.</a> Poult Sci. 74 (2), 374-382 (1995).</li><li>Ding, S. T., Lilburn, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+expression+of+acyl-coenzyme+A:+cholesterol+acyltransferase+in+the+yolk+sac+membrane,+liver,+and+intestine+of+developing+embryos+and+posthatch+turkeys.">The developmental expression of acyl-coenzyme A: cholesterol acyltransferase in the yolk sac membrane, liver, and intestine of developing embryos and posthatch turkeys.</a> Poult Sci. 79 (10), 1460-1464 (2000).</li><li>Ding, S. T., Lilburn, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ontogeny+of+fatty+acid-binding+protein+in+turkey+(Meleagridis+gallopavo)+intestine+and+yolk+sac+membrane+during+embryonic+and+early+posthatch+development.">The ontogeny of fatty acid-binding protein in turkey (Meleagridis gallopavo) intestine and yolk sac membrane during embryonic and early posthatch development.</a> Poult Sci. 81 (7), 1065-1070 (2002).</li><li>Kanai, M., Soji, T., Sugawara, E., Watari, N., Oguchi, H., Matsubara, M., Herbert, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Participation+of+endodermal+epithelial+cells+on+the+synthesis+of+plasma+LDL+and+HDL+in+the+chick+yolk+sac.">Participation of endodermal epithelial cells on the synthesis of plasma LDL and HDL in the chick yolk sac.</a> Microsc Res Tech. 35 (4), 340-348 (1996).</li><li>Mobbs, I. G., McMillan, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+endodermal+epithelium+of+the+chick+yolk+sac+during+early+stages+of+development.">Structure of the endodermal epithelium of the chick yolk sac during early stages of development.</a> Am J Anat. 155 (3), 287-309 (1979).</li><li>Mobbs, I. G., McMillan, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+across+endodermal+cells+of+the+chick+yolk+sac+during+early+stages+of+development.">Transport across endodermal cells of the chick yolk sac during early stages of development.</a> Am J Anat. 160 (3), 285-308 (1981).</li><li>Nakazawa, F., Alev, C., Jakt, L. M., Sheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yolk+sac+endoderm+is+the+major+source+of+serum+proteins+and+lipids+and+is+involved+in+the+regulation+of+vascular+integrity+in+early+chick+development.">Yolk sac endoderm is the major source of serum proteins and lipids and is involved in the regulation of vascular integrity in early chick development.</a> Dev Dyn. 240 (8), 2002-2010 (2011).</li><li>Noble, R. C., Cocchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+metabolism+and+the+neonatal+chicken.">Lipid metabolism and the neonatal chicken.</a> Prog Lipid Res. 29 (2), 107-140 (1990).</li><li>Shand, J. H., West, D. W., McCartney, R. J., Noble, R. C., Speake, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+esterification+of+cholesterol+in+the+yolk+sac+membrane+of+the+chick+embryo.">The esterification of cholesterol in the yolk sac membrane of the chick embryo.</a> Lipids. 28 (7), 621-625 (1993).</li><li>Speier, J. S., Yadgary, L., Uni, Z., Wong, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+of+nutrient+transporters+and+digestive+enzymes+in+the+yolk+sac+membrane+and+small+intestine+of+the+developing+embryonic+chick.">Gene expression of nutrient transporters and digestive enzymes in the yolk sac membrane and small intestine of the developing embryonic chick.</a> Poult Sci. 91 (8), 1941-1949 (2012).</li><li>Walzem, R. L., Hansen, R. J., Williams, D. L., Hamilton, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+Induction+of+VLDLy+Assembly+in+Egg-Laying+Hens.">Estrogen Induction of VLDLy Assembly in Egg-Laying Hens.</a> J Nutr. 129 (2), 467S-472S (1999).</li><li>Yoshizaki, N., Soga, M., Ito, Y., Mao, K. M., Sultana, F., Yonezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+consumption+of+yolk+granules+during+the+development+of+quail+embryos.">Two-step consumption of yolk granules during the development of quail embryos.</a> Dev Growth Differ. 46 (3), 229-238 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> alamarBlue Cell Viability Assay Protocol. , (2008).</li><li>Lin, H. Y., Chen, C. C., Chen, Y. J., Lin, Y. Y., Mersmann, H. J., Ding, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Amelioration+of+High-Fat+Diet-Induced+Fatty+Liver+by+Docosahexaenoic+Acid+and+Lysine+Supplementations.">Enhanced Amelioration of High-Fat Diet-Induced Fatty Liver by Docosahexaenoic Acid and Lysine Supplementations.</a> Biomed Res Int. 2014 (1), 310981 (2014).</li><li>Lin, Y. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adiponectin+receptor+1+regulates+bone+formation+and+osteoblast+differentiation+by+GSK-3&#946;/&#946;-Catenin+signaling+in+mice.">Adiponectin receptor 1 regulates bone formation and osteoblast differentiation by GSK-3&#946;/&#946;-Catenin signaling in mice.</a> Bone. 64, 147-154 (2014).</li></ol>

There is no competing financial interests or other conflicts of interest. We did not get any funding from any private company.

因为以前的培养系统只有有限的成功，需要一个更好的培养系统。日本鹌鹑YSM内胚层需要与蛋白水解酶如胶原酶松开细胞 - 细胞连接以实现在离体的外植体更好的生长性能的治疗。我们的数据显示，从局部消化处理的细胞数接种2天（ 图1）后，比从未消化的组织培养更大。因此，部分水解消化是改善从YSM细胞产量的关键步骤。 在当前的文化协议的关键修改是部分...

禽流胚胎的主要营养资源是蛋黄，33％脂质，17％的蛋白质组成，和1％的灰分。1在胚胎发育期间，卵黄囊膜（YSM）从胚胎腹腔内生长并逐渐覆盖蛋黄表面。在2天胚胎开始，与脂质代谢和血管发生相关的基因的表达在YSM逐渐增加，并且YSM发展缓慢绒毛状突起。-8,9-这些突起增加蛋黄营养的吸收，以支持胚胎发育。该YSM是包含三个胚层，内胚层，中胚层和外胚层的胚外组织。14卵黄囊外胚层面临的蛋白和链接与卵黄膜轻轻地覆盖卵黄囊。内胚层上皮细胞直接面对朝蛋黄作为养分利用率的门户网站。6作为绝经年限的扩大，内胚层上皮细胞（EECS）可以通过十八分e和功能分为两组，区卵黄和面积vasculosa 7 区卵黄是由内胚层细胞是从胚胎遥远;区域vasculosa是由胚层细胞，并覆盖有血管和结缔组织有区别的EECS。通过5天的胚胎，蛋黄完全由YSM的外胚层和内胚层覆盖，血管面积增长迅速。在YSM吸收，重新组成并释放脂质（如蛋黄衍生的极低密度脂蛋白）和蛋白质进入胚胎循环系统9中，2。因此，我们建立了主日本鹌鹑胚胎内胚层上皮细胞培养系统，来研究脂质的机制利用率YSM禽流胚胎发育过程中。 脂质如甘油三酯，磷脂，磷脂和胆固醇酯（CE）是禽类胚胎的主要能源。在开发的早期阶段，蛋黄脂类是Composed只有1.3％，CE和它上升到10-15％的鸟类胚胎发育的3期中，11胆固醇酯是由胆固醇在鸡胚YSM固醇O-酰基转移酶1（SOAT1）合成。4 胆固醇的存储形式是CE，CE是脂蛋白携带和脂蛋白被循环输送到组织中。13一个星期前舱门有鸟类胚胎的快速增长。大约在蛋黄中剩余的脂肪含量68％，在这一阶段被吸收。10由蛋黄脂质利用可通过EEC研究模型加以澄清机制。阿鸡欧洲经济共同体培养协议的建立是为了实现这一研究的目标。2,9然而，由于组织外植体的成功率很低，需要一种改进的欧洲经济共同体的细胞培养过程来研究某些酶和蛋白质的功能在通过介导养分的利用在开发过程中禽流感的胚胎。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco by Life technologies</td><td>12800-017 10 X 1 L</td><td>For wash the EECs pellets</td></tr><tr><td>D-MEM/F-12</td><td>Gibco by Life technologies</td><td>12400-024 10 X 1 L</td><td>As the basal medium in culturing EECs</td></tr><tr><td>NBCS</td><td>Gibco by Life technologies</td><td>16010-159</td><td>As the supplyment serum in culturing EECs</td></tr><tr><td>Pen-Strep Ampho. Solution</td><td>BI (Biological Industries)</td><td>03-033-1B 100 ml</td><td>For attenuating the possible infection&amp;nbsp;</td></tr><tr><td>Collagenase Type IV</td><td>Gibco by Life technologies</td><td>17104-019 1 g</td><td>&amp;nbsp;Collagenase is a protease with specificity for the bond between a neutral amino acid (X) and glycine in the sequence Pro-XGly-Pro. As the protease for dissociation of cells from primary tissue.</td></tr><tr><td>24-well plate</td><td>FALCON&lt;sup&gt;&amp;reg;&lt;/sup&gt;</td><td>REF-353047</td><td>For EECs to attach and extension</td></tr><tr><td>50 ml PP centrifuge tubes</td><td>Corning&lt;sup&gt;&amp;reg;&lt;/sup&gt; CentriStar&lt;sup&gt;TM&lt;/sup&gt;</td><td>430829</td><td>For transportion of membranes and enzyme digestion</td></tr><tr><td>50 ml Conical bottomed Tube with Cap</td><td>PRO TECH</td><td>CT-50-PL-TW</td><td>For transportion of membranes and enzyme digestion</td></tr><tr><td>Reciprocal shaking bath</td><td>DEAGLE</td><td>SB302</td><td>For better enzymatic digestion on membranes</td></tr></tbody></table>

primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos

我们建立了一个内胚层上皮细胞培养模型（EEC）用于研究开发过程中由禽胚胎调解养分利用某些酶和蛋白质的功能。受精日本鹌鹑蛋在37℃下培养5天，然后卵黄囊膜（YSM）收集来建立欧洲经济共同体培养系统。我们从YSM分离胚胎内胚层层，和切片膜进入2 - 3毫米件和在24孔培养板播种前用胶原酶部分消化。该EECS繁殖出来的组织和准备​​好细胞培养研究。我们发现，EECS有体内 YSM的典型特征，例如，脂滴的累积，固醇O-酰基转移酶和脂蛋白脂肪酶的表达。部分消化处理显著上升EEC文化的成功率。利用EECS，我们证明了SOAT1的表达受cAMP的调节Ðependent蛋白激酶相关途径。这主要日本鹌鹑EEC培养体系是研究胚胎脂质运输和澄清参与禽流感在胚胎发育过程中的调解YSM养分的利用基因的作用的有用工具。

为了实现建立一个一致的和有用的细胞模型的目标，我们需要扩大和稳定增殖率和禽流EECS的性能。我们比较了没有酶消化与内胚层与蛋白水解酶，如胶原酶或胶原酶加分散酶的0.6单位部分消化的内胚层的直接温育。分散酶是一种氨基肽酶是水解的非极性氨基酸残基的N-末端肽键。而没有消化（ 图1）相比，蛋白酶消化处理增加的细胞增殖，细胞生长不五天后孵...

Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

日本鹌鹑胚胎的卵黄囊膜主内胚上皮细胞培养

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

primary-endodermal-epithelial-cell-culture-from-yolk-sac-membrane

Research

JoVE Journal

Developmental Biology

8.8K 次观看.  National Taiwan University. To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

视频: Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

Method for Culture of Early Chick Embryos ex vivo (New Culture)

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 patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

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

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the formation of quail-chicken chimeras and the use of the chorioallantoic membrane (CAM) to sustain the development of ectopic tissues date back to the last century. Nowadays, the combination of these classical techniques with recent in vitro methodologies offers novel prospects to further explore organ formation.
Here we describe a two-step approach to study early- and late-stages of organogenesis. Briefly, the embryonic region containing the presumptive territory of the organ is isolated from quail embryos and grown in vitro in an organotypic system (up to 48 h). Cultured tissues are subsequently grafted onto the CAM of a chicken embryo. After 10 days of in ovo development, fully formed organs are obtained from grafted tissues. This method also allows the modulation of signaling pathways by the regular administration of pharmacological agents and tissue genetic manipulation throughout in vitro and in ovo developmental steps. Additionally, developing tissues can be collected at any time-window to analyze their gene-expression profile (using quantitative PCR (qPCR), microarrays, etc.) and morphology (assessed with conventional histology and immunochemistry).
The described experimental procedure can be used as a tool to follow organ formation outside the avian embryo, from the early stages of organogenesis to fully formed and functional organs.

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

鸟类模型中器官形成早期和晚期的两步探讨: 胸腺和甲状旁腺器官发生范式

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

发育生物学

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

脊髓培养法研究胚胎鸡组织的发展

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties make it an excellent in vivo experimental model to study angiogenesis, tumor growth, drug delivery systems, or photodynamic diagnosis (PDD) and photodynamic therapy (PDT). At the same time, this model addresses the requirement for the replacement of experimental animals with a suitable alternative. Ex ovo cultivated embryo allows easy substance application, access, monitoring, and documentation. The most frequently used is chick CAM; however, this article describes the advantages of the Japanese quail CAM as a low-cost and high-throughput model. Another advantage is the shorter embryonic development, which allows higher experimental turnover. The suitability of quail CAM for PDD and PDT of cancer and microbial infections is explored here. As an example, the use of the photosensitizer hypericin in combination with lipoproteins or nanoparticles as a delivery system is described. The damage score from images in white light and changes in fluorescence intensity of the CAM tissue under violet light (405 nm) was determined, together with analysis of histological sections. The quail CAM clearly showed the effect of PDT on the vasculature and tissue. Moreover, changes like capillary hemorrhage, thrombosis, lysis of small vessels, and bleeding of larger vessels could be observed. Japanese quail CAM is a promising in vivo model for photodynamic diagnosis and therapy research, with applications in studies of tumor angiogenesis, as well as antivascular and antimicrobial therapy.

The chorioallantoic membrane (CAM) of the avian embryo is a very useful and applicable tool for various areas of research. A special ex ovo model of Japanese quail CAM is suitable for photodynamic treatment investigation.

鹌鹑绒毛膜尿囊膜 - 光动力诊断和治疗的工具

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties ...

癌症研究

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

胸腺标本分离和鸡刺激器官的形成

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that the amniotic epithelium is a homogeneous layer. The human amnion can be divided into three anatomical regions: reflected, placental, and umbilical. Each region has different physiological roles, such as in pathological conditions. Here, we describe a protocol to dissect human amnion tissue in three sections and maintain it in vitro. In culture, cells derived from the reflected amnion displayed a cuboidal morphology, while cells from both placental and umbilical regions were squamous. Nonetheless, all the cells obtained have an epithelial phenotype, demonstrated by the immunodetection of E-cadherin. Thus, because the placental and reflected regions in situ differ in cellular components and molecular functions, it may be necessary for in vitro studies to consider these differences, because they could have physiological implications for the use of HAEC in biomedical research and the promising application of these cells in regenerative medicine.

This protocol describes the isolation of epithelial cells from different anatomical regions of the human amniotic membrane to determine their heterogeneity and functional properties for possible application in clinical and physiopathological models.

人类羊膜不同解剖区域的皮下细胞体外培养

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

一个 EX - OVO鸡胚胎培养系统适用于成像和显微外科的应用

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

禽流感胚胎瓣膜祖细胞的分离和培养

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

鸡颅神经嵴细胞培养物的制备及形态学分析

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...