Name: studying wnt signaling during patterning of conducting airways
Uploaded: 2016-10-16T13:00:00.000+00:00
Duration: 13 min
Description: Watch this Scientific Journal Video about Studying Wnt Signaling During Patterning of Conducting Airways at JoVE.com

我们承认麦克Muntifering和马特Kofron激光共聚焦成像和盖尔马科与组织程序的帮助。这项工作是部分由卫生NHLBI全国学院（K01HL115447到DS）的支持。

动物饲养在无病原体的条件。小鼠根据由CCHMC机构动物护理和使用委员会（俄亥俄州辛辛那提美国）批准的方案进行处理。在这些研究中使用的小鼠保持在混合的背景。 1.全山的X半乳糖苷酶染色<ol><li>安乐死怀孕的女性在E11.5至E14.5，用CO 2吸入。放置的动物在CO 2室中，该腔室用CO 2进行充电。保持在腔室的动物为5分​​钟最小值。颈椎脱位安乐死进行二次方法。 </li><li>用乙醇（EtOH中）的70％，并用单个腹部中线切口进行剖腹清洁腹部区域。利用手术剪和标准模式（直锯齿）钳。 </li><li>在培养皿隔离使用镊子和剪刀精子宫角和立即将组织冷冻含有PBS液。保持在冰上，直到程序组织胚胎的隔离。 </li><li>隔离子宫角的胚胎。使用解剖显微镜解剖胚胎气管肺组织。利用精尖镊子和剪刀持有和刺穿子宫组织和concepti释放胚胎。 <ol><li>除去剩余的胚胎膜。随着胚胎的针刀片切援助头部和膈下下半身的一部分。找到肝脏作为一个标志性建筑。 </li><li>胚胎的胸部区域的隔离后，继续清除体内壁，脊椎和心脏用针叶片的侧面。同时消除的心脏，以避免气管组织损伤照顾。最后，细心，用针叶片援助拉动食道分开气管食道。保持冰PBS液组织。 </li></ol></li><li>在4毫升地方组织螺丝用玻璃或移液管口小瓶。固定气管肺组织在PBS中的4％多聚甲醛（PFA）30分钟。固定后，组织冲洗用PBS。虽然漂洗，准备的X-gal染色溶液（加上5 mm K 3的Fe（CN）6，（100微升0.5M的股票）5毫米K 4的Fe（CN）6（100微升0.5M的股票）2毫米氯化镁2，（ 20微升1M的股票）0.01％NaDOC（50微升2％的股份），0.02％NP 4 O（100微升2％的股票），1毫克/毫升的X-gal（500微升20毫克/毫升股票）和9.13毫升的蒸馏水，使至10ml的终体积）。 </li><li>除去任何剩余的PBS中，并加入2毫升X-gal的染色液到玻璃小瓶中。将小瓶在摇床托盘上。染色组织1〜2小时中的X-gal染色溶液。孵育的X-gal溶液中的组织为4小时，以便进一步处理和嵌入。 </li><li>停止通过洗涤组织在3％二甲基亚砜-PBS中进行反应。冲洗在PBS，3次，每次5分钟，在70％乙醇各冲洗和存储。 </li><li>填培养皿1％琼脂糖/ PBS溶液。允许琼脂糖巩固。用玻璃移液管，将琼脂糖合作组织ated板充满了PBS的解决方案。照片用解剖显微镜整个坐骑。选择明适当的过滤器。 </li><li>为了更好地为区分石蜡包埋和切片的X-gal染色过程的样本点。将样品在细筛纸盒。 <ol><li>要处理的石蜡包埋标本使用自动化的处理器，并成立了短周期如下:酒精的六个转变:5分钟，第一个变化，每其余每个变3分钟。二甲苯的三个转变:5分钟，第一个变化，3分钟接下来的两个变化。在30℃下执行前面的步骤。最后，执行在62℃的石蜡的三种变化:25分钟，8分钟和5分钟。 </li></ol></li><li>东方样品与组织平趴在嵌入船的底部嵌入。小心加入石蜡以填充嵌入舟。立即清凉嵌入站，直到石蜡凝固的冷板。从嵌入船上卸下块。使用切片机，生成6微米小号ections。在预处理 - 幻灯片和投影片温暖定在42℃1小时干片山路段。 </li><li>烘烤炉中在56ºC幻灯片过夜。将幻灯片机架。 Deparaffinize滑动用100％二甲苯的三种变化，每次10分钟。使用染色填充用200毫升二甲苯的菜肴。 </li><li>用核固红复染为10至30秒之前再水化使用梯度乙醇系列（100％，70％，50％和30％，每步1分钟），以PBS中的幻灯片。冲洗载玻片用自来水对每个时间以除去过量的核固红的5分钟三次。 </li><li>脱水在二甲苯的三种变化洗涤之前，在分级乙醇一系列幻灯片（50％，70％和100％EtOH中）（20骤降每变化）和盖与基于二甲苯安装媒体打滑。 </li></ol> 2.凝集素染色<ol><li>如步骤1.1至1.4中所述解剖E13.5和E14.5气管肺组织。在螺丝帽小瓶用玻璃移液管处组织。固定气管肺组织用2ml2％PFA溶液过夜，在4℃。固定后，冲洗样品在PBS中持续10分钟，每次洗涤三次。 </li><li>制备封闭缓冲液，通常为10毫升的溶液的最终体积。 0.1克BSA（1％）添加至8毫升的PBS。允许BSA溶解。增加±ml山羊血清（2％）的Triton X-100（0.3％）的0.03毫升的。带来最终体积至10ml，用PBS。块样品在0.5ml在室温下封闭缓冲液1小时。 </li><li>使用移液管除去封闭缓冲液，并用由50微克/微升外源凝集素，10％山羊血清和PBS的凝集素溶液替换。用250微升每个样品凝集素的解决方案。在4℃孵育过夜。确保覆盖含铝箔组织玻璃瓶。用外源凝集素溶液温育后，冲洗样品在PBS三次10分钟。 </li><li>地方外植体在含有足够的PBS以覆盖样品的琼脂糖包被的培养皿。照片使用荧光解剖显微镜整个坐骑。选择appropriate过滤器来检测荧光。凝集素PNA通常被耦合到GFP。 </li></ol> 3.全山免疫染色和共聚焦显微镜<ol><li>隔离气管肺组织，转移至4毫升的玻璃螺旋盖小瓶中并在4％PFA固定过夜。 E11.5组织可以固定在2％的PFA。贮存于甲醇100％。样品可在-20℃保存长达几个月。 </li><li>使用移液管除去甲醇并用登特的漂白（4份甲醇，1份DMSO和1份30％的H 2 O 2）2小时通透样品。水合物组织在分级系列甲醇的PBS稀释如下:100％甲醇，75％甲醇，50％甲醇，25％甲醇，100％PBS中。孵育期间水合的每一步在室温下10分钟。 </li><li>块组织在PBS中稀释的0.5％封闭试剂（详见材料 ），在室温下搅拌两小时。 </li><li>稀释主要抗体阻断溶液（Sox9的1:100，Nkx2.1 1:200，αSMA1:250），并在4℃下过夜孵育样品。洗涤样品在室温下用PBS中的五倍。 1小时执行每洗。 </li><li> 500稀释0.5％封闭液:在1应用二次抗体。仔细选择的二抗，防止二次抗体之间的结合。在黑暗的房间在4℃孵育过夜。洗涤样品，在室温下20分钟三次。脱水样品在分级系列甲醇的PBS稀释如下:25％甲醇，50％甲醇75％甲醇，100％甲醇，10分钟的每个步骤。 注意:确保样品保持覆盖铝箔，并尽量减少曝光。组织现在可以储存在4℃下几个星期，直到拍照。 </li><li>转移样品定制的舞台板，除去甲醇和清晰的大约200微升穆雷明确的解决方案19,20（2份苯甲酸苄酯，1份苯甲醇）立即BEF矿石成像。照片使用共聚焦显微镜。处理和使用图像处理软件分析图像。 </li></ol> 4.细胞增殖<ol><li>注入E11.5妊娠小鼠腹膜内与100微克的BrdU / g体重的浓度的BrdU溶液。截至E12.5 E13.5或在协议第1条所述并隔离胚胎牺牲女性。使用刀片，消费头和下方胸腔身体的下部。 </li><li>放置胸椎组织在4毫升螺旋盖小瓶中并在1毫升4％的PFA固定过夜。 30％，50％和70％的乙醇，5分钟的每个步骤:洗涤样品用PBS两次5分钟，并通过乙醇系列中蒸馏水稀释至70％的乙醇如下脱水样品。 <ol><li>将样品在中等大小的屏幕录像带。流程组织进行石蜡包埋使用和自动化处理器。设置一个周期如下:8分钟每醇六变化，6分钟每个，三C二甲苯的三种变化石蜡hanges，25分钟，9分钟，8分钟。 </li></ol></li><li>嵌入石蜡定向在希望的位置的组织为在载玻片上在部分1.将部分所述生成6微米的横向或纵向截面，并允许组织附着1小时在42℃上滑动温暖集滑动。 </li><li>烘烤的幻灯片，在其两侧，一夜之间在56℃的烤箱。放在塑料衣架幻灯片。在100％二甲苯三个变化，每变化10分钟浸泡德paraffinize幻灯片。可使用200毫升二甲苯的完全淹没幻灯片。水化使用梯度乙醇系列（100％，70％，50％和30％，每步5分钟搅拌），以PBS（5分钟）的幻灯片。 </li><li>执行使用10mM柠檬酸缓冲液，pH 6。要制备100ml柠檬酸盐缓冲液混合18毫升的0.1M的柠檬酸一水合物和82毫升0.1N柠檬酸钠的抗原修复。 <ol><li>将样品在充满抗原检索解决方案和微波炉在高功率6.5分钟塑料罐子科普林。添加DIS耕种水科普林缸40％的电力，以补偿蒸发的柠檬酸缓冲液和再热6分钟。 </li><li>填科普林缸与水到顶部，并在40％的功率再次再加热6分钟。微波时间可能会因使用的微波炉。 </li><li>允许滑动到在室温下洗涤载玻片之前在蒸馏水中冷却20分钟，1分钟，接着PBS 5分钟。与PBS洗净后，块载玻片2小时在封闭含有的TBS溶液（2.425克Tris碱的和在1升的蒸馏水稀释8.765克的NaCl）10％驴血清和1％BSA。 </li></ol></li><li>加入阻断溶液（含有10％驴血清和1％BSA的TBS）稀释的初级抗体。的BrdU（有丝分裂标志物），Nkx2.1（呼吸道上皮），Sox9的（细胞会引起软骨）和αSMA（平滑肌细胞）。孵育的幻灯片过夜，在4℃。使用覆盖方法以节约抗体。应用180微升抗体与一个垫圈，然后附加滑动仿佛盖玻片平。 </li><li>通过浸泡滑入蒸馏水分离垫片。除去垫片后，通过用含有0.1％吐温-20的TBS进行6 5分钟洗涤洗涤未结合的初级抗体。 <ol><li>孵育载玻片在以1:1的稀释封闭液稀释的二级抗体:200，在室温下1小时。精心选择的二抗，以防止它们之间不需要的绑定。通过用含有0.1％Tween的TBS进行三五分钟的清洗除去未结合的次级抗体。 </li><li>洗在0.1M Tris碱载玻片两次5分钟，然后0.05M的Tris碱两次5分钟。幻灯片可能在0.05M Tris碱留在使用安装介质具有或不具有DAPI（1.5微克/毫升）coversliping。在幻灯片文件夹中存储的幻灯片在4°C和覆盖文件夹用铝箔避光。 </li></ol></li><li>可视化染色和使用自动荧光显微镜照片。算在拍摄标记细胞和每场的总细胞20X和40X以确定增殖细胞对总细胞的比率。 </li></ol>

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U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-reconstruction+of+blood+vessels+by+ultramicroscopy.">3D-reconstruction of blood vessels by ultramicroscopy.</a> Organogenesis. 5, 145-148 (2009).</li><li>Park, J., 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=Regulation+of+Sox9+by+Sonic+Hedgehog+(Shh)+is+essential+for+patterning+and+formation+of+tracheal+cartilage.">Regulation of Sox9 by Sonic Hedgehog (Shh) is essential for patterning and formation of tracheal cartilage.</a> Dev Dyn. 239, 514-526 (2010).</li><li>Elluru, R. G., Thompson, F., Reece, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+18+gives+growth+and+directional+cues+to+airway+cartilage.">Fibroblast growth factor 18 gives growth and directional cues to airway cartilage.</a> Laryngoscope. 119, 1153-1165 (2009).</li><li>Ahnfelt-Ronne, J., 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=An+improved+method+for+three-dimensional+reconstruction+of+protein+expression+patterns+in+intact+mouse+and+chicken+embryos+and+organs.">An improved method for three-dimensional reconstruction of protein expression patterns in intact mouse and chicken embryos and organs.</a> J Histochem Cytochem. 55, 925-930 (2007).</li><li>Yang, B., 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=Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158, 945-958 (2014).</li><li>Gillotte, D. 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"作者什么都没有透露。"

事件呼吸道的形态底层不完全了解，特别是用于传导气道的图案化所需的过程。先前的研究已利用离体技术，其特征在于显影外植体在空气-液体相间培养或嵌入在基质胶21,22。这些研究已经表明生长因子如何影响显影气管的构图和气管软骨的形成。一个限制到这些研究是该组织的结构没有正确保持，因此，他们可能不太概括软骨的体内过程。 图案形成列入加上整装染色技术，免疫荧光成像和体内研究，我们进行了研究Wnt信号的传导气道的过程中的作用的方法。其次是本身整装组织的X-gal染色ctioning和反染色，提出过石蜡切片的染色的优点。整装染色可以直接检测网站，Wnt信号/β-catenin的活动是可操作的。此外，染色外植体切片确定在间质，其中的活性定位为发展进步的精确地点。后者比检测石蜡切片β半乳糖苷酶的表达的更好和准确的读出。而酶活性可在部分被检测，此过程将需要冷冻切片，将不总是容易产生，并且可以不保留小样本的体系结构。在本文中描述的协议的限制，为整装染色后石蜡包埋的样品的处理，会造成信号的丢失。因此，样品需要在X-gal的溶液保持更长和石蜡包埋较短周期应该被用来在部分得到良好的信号。 另一方面，整装免疫荧光法，通过Ahnfelt-龙尼23记载的技术后修饰，允许显影气管和肺组织的三维可视化，与由关键转录因子如Nkx2.1和编码的蛋白质的一个限定的表达模式SOX9。当需要时，整个安装染色可以呈现为图像的共焦辅助Z图库，描绘了气管肺组织的不同深度。而成像小外植体，组织的清除应该在一个舞台板来执行。我们利用厚的金属其中，样品被清零，成像定制光学阶段性底部板。这可以防止材料的损失，同时清除步骤之后转印小组织。虽然整装免疫是小型组织染色合适的技术，我们认识到技术局限。特别是，我们观察到更好地识别抗原中的部分，而不是整个装载的抗体的异种表演。如果没有备用的抗体可用此问题可能妨碍该技术的性能。它也有可能是与Murray的明确使用甲醇和结算的可以具有在观察到的信号产生负面影响。在未来，我们将测试其他非有机结算解决方案，并适当调整协议，为此24。最后需要考虑的是，整装将允许在同一给定的时间在一个组织中表达一些蛋白质的研究。 以前的报告显示的用处PNA凝集素染色检测气管组织21,25的部分间质凝聚。在本研究中，我们报告由在整装，这有利于气管的区域的更好的可视化，其中所述的软骨将形成进行染色的方法。在执行外源凝集素染色护理应放置在使用凝集素的适当的浓度为不超过染色，作为市售的PNA-凝集素结合到GFP。组织的自发荧光可能掩盖的结果使得难以分辨对应的间充质凝聚带。 最后， 在体内其次切片和免疫荧光染色用BrdU组织的标记是一种可行的技术来确定呈现了典型的和静态的染色，如PPH 3或PCNA 26,27优点细胞增殖。后者的技术提出的限制，如染色将根据变化细胞周期阶段。这个问题是由我们所描述的技术消除。组合体内标记随后包埋，切片和免疫荧光使我们能够确定上皮的Wnt配体平衡的Sox9和α-SMA的表达细胞，这将引起气管软骨和肌肉7差动增殖。 总之，多技术的方法来研究的显影呼吸道的图案形成允许Wnt信号在传导气道形态的作用的深入分析。这些研究确定的Wnt /β-catenin在气管组织的精确位点，以及由该所需的规格，气管间质细胞谱系的差扩散，以及软骨形成的气管上皮提供感性和启发性信令。我们未来的方向包括整个安装染色为最近发现WLS优化靶基因，以及与外源凝集素的染色组合执行整装免疫荧光。

呼吸道发展是通过用Nkx2.1阳性细胞中的腹侧内胚层前肠1,2-外观胚胎9天（E9）来启动。食管-气管套管分离将E11.5解决将管可以区分为不同的实体，分别由间质组织3所包围。 Wnt信号起着呼吸道作为WNT2和WNT2B缺失的说明书中的关键作用，通过从内胚呼吸道上皮内脏间质和β连环蛋白的缺失表达将导致肺发育不全4,5。我们先前的研究确定删除WLS，所有的Wnt配体的受体货物介导的分泌，从肺发育不良的内胚呼吸道结果，肺血管发展和气管间质6,7的错误图案的缺陷。这些数据支持这样的上皮 - 间质CRO的重要性SS交谈在细胞分化和说明书中，因为它也已在其它研究8,9所示。 肺发育的最初阶段的研究依赖于遗传， 在使我们能够更好地理解机制驾驶呼吸身份10-16 体外和体外技术。在空气液体相间全肺外植体培养已被广泛用来研究肺分支形态10,17,18的早期阶段的生长因子的作用。虽然这种方法被用作形态学变化，如分支形态和基因表达的调制读出，它是有限的发育过程的早期阶段的研究中，作为培养本身不支持脉管17的发展。气管软骨的发展需要更长的孵化时间，可能与此培养技术不兼容。 要analyzÈ呼吸道形成过程中的Wnt信号传导的作用，我们已经适应的标准技术，以满足我们的胚胎的研究的需要。我们已经修改卷，染色次，石蜡包埋和定时气管，肺组织清算处理循环。优化在本研究中所描述的技术的主要目的是分析气管发展的早期阶段中所发生从E11到E14.5小鼠。使用报告小鼠线Axin2LacZ在发展中国家间质气管的Wnt我们/β-catenin的活动的精确确定地点。我们还适合整装气管组织凝集素染色的过程。因此，我们能够想象的间充质凝结和预测网站，软骨会发生。整装和WlsShhCre小鼠，加上先进的显微技术获得的胚胎组织切片，染色使我们能够推出由TRA所产生的Wnt配体的作用CHEAL上皮气管图案。

<table><tbody><tr><td>Anti Sox9 ab.</td><td>Millipore</td><td>AB5535</td><td>1:400 , rabbit</td></tr><tr><td>Anti Sox9 ab.</td><td>Santa Cruz</td><td>Sc-20095</td><td>1:50, rabbit</td></tr><tr><td>Anti Smooth Muscle Actin ab.</td><td>Sigma</td><td>A5228</td><td>1:2k, mouse</td></tr><tr><td>Anti NKX2.1 ab.</td><td>Seven Hills</td><td>n/a</td><td>1:100, guinea pig</td></tr><tr><td>Anti NKX2.1 ab.</td><td>Seven Hills</td><td>n/a</td><td>1:400, mouse</td></tr><tr><td>Anti Brdu ab.</td><td>Abcam</td><td>AB1893</td><td>1:200, sheep</td></tr><tr><td>Anti Brdu ab.</td><td>Santa Cruz</td><td>Sc-32323</td><td>1:4k, mouse</td></tr><tr><td>PNA Lectin</td><td>Sigma</td><td>L 7381</td><td></td></tr><tr><td>Secondary antibodies</td><td>Life technologies</td><td></td><td>Alexa fluor Molecular probes</td></tr><tr><td>K&lt;sub&gt;3&lt;/sub&gt;Fe(CN)&lt;sub&gt;6&lt;/sub&gt;</td><td>Sigma</td><td>P8131</td><td></td></tr><tr><td>K&lt;sub&gt;4&lt;/sub&gt;Fe(CN)&lt;sub&gt;6&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P3289</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M9272</td><td></td></tr><tr><td>NaDOC</td><td>Life Technologies</td><td>89905</td><td></td></tr><tr><td>NP&lt;sub&gt;4&lt;/sub&gt;O</td><td>Life Technologies</td><td>85124</td><td></td></tr><tr><td>Alcian Blue 8GX</td><td>Sigma</td><td>A-3157</td><td></td></tr><tr><td>Fisher brand super-frost plus</td><td>Fisher</td><td>12-550-15</td><td></td></tr><tr><td>PFA (16%)</td><td>EMS</td><td>15710</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>70011-044</td><td></td></tr><tr><td>Fetal Calf Serum</td><td>Sigma</td><td>11K413</td><td></td></tr><tr><td>Blocking reagent</td><td>Invitrogen</td><td></td><td>Component of TSA kit #2&amp;nbsp;&amp;nbsp;&amp;nbsp; ( T20932)</td></tr><tr><td>BrDu</td><td>Sigma</td><td>B5002-5g</td><td></td></tr><tr><td>Vectashield mounting medium</td><td>Vector labs</td><td>H-1000</td><td></td></tr><tr><td>Permount</td><td>Fisher</td><td>SP15-500</td><td></td></tr><tr><td>Tissue-loc cassettes Histoscreen</td><td>Fisher</td><td>C-0250-GR</td><td></td></tr><tr><td>Biopsy cassettes</td><td>Premiere</td><td>BC0109</td><td>Available in different colors</td></tr><tr><td>Nuclear fast red&amp;nbsp; Kernechtrot 0.1%</td><td>Sigma</td><td>N3020</td><td></td></tr><tr><td>Citric acid</td><td>Sigma</td><td>C1909-500G</td><td></td></tr><tr><td>Sodium citrate tribasic dihydrate</td><td>Sigma</td><td>S4641-1Kg</td><td></td></tr><tr><td>Trizma hydrochloride</td><td>Sigma</td><td>T5941-500G</td><td></td></tr><tr><td>Xylene</td><td>Pharmco-AAPER</td><td>399000000</td><td></td></tr><tr><td>Ethanol</td><td>Pharmco-AAPER</td><td>111000200</td><td></td></tr><tr><td>Micro knives</td><td>FST</td><td>10318-14</td><td></td></tr><tr><td>Dumont #5 ceramic coated</td><td>FST</td><td>11252-50</td><td></td></tr><tr><td>Dumont #5CO</td><td>FST</td><td>11295-20</td><td></td></tr><tr><td>Dumont # 5</td><td>FST</td><td>91150-20</td><td></td></tr><tr><td>Thermo/Shandon Excelsior ES</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>RM2135</td><td></td></tr><tr><td>Nikon i90</td><td>Nikon</td><td></td><td>Wide field microscope</td></tr><tr><td>NikonA1Rsi</td><td>Nikon</td><td></td><td>Confocal microscopy. Settings:NikonA1 plus camera, scanner: Galvano, detector:DU4. Optics Plan Apo lambda 10X. Modality: Widefield fluorescence laser confocal.&amp;nbsp;</td></tr><tr><td>Leica MS 16 FA</td><td>Leica</td><td></td><td>Fluorescence Dissecting microscope</td></tr><tr><td>Zeiss</td><td>Zeiss</td><td></td><td>Automated fluorescence microscope</td></tr><tr><td>Leica Application suite</td><td>Leica</td><td></td><td>Leica imaging software</td></tr><tr><td>NIS</td><td>Nikon</td><td></td><td>Nikon imaging software</td></tr><tr><td>IMARIS</td><td>Bitplane</td><td></td><td>Imaging processing software</td></tr></tbody></table>

studying wnt signaling during patterning of conducting airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

的Wnt /β-catenin的活性从记者紫胶的Axin2小鼠-Z分离出11胚胎气管，肺组织中检测到整装紫胶-Z染色。染色网站注明的Wnt /β-catenin的活动。整装染色的切片的分析中确定的Wnt /β-catenin的活性存在于气管的间质和在显影肺部周边区域的间质。在WlsShhCre胚胎（其中，从呼吸道的上皮的Wnt配体的分泌废除）紫胶-Z的染色几乎不存在（ 图1）。 在气管间充质质凝聚在软骨的一个关键步骤是由间质细胞注定给出r过程ISE软骨凝结。这些细胞紧密聚集被称为间充质凝聚。为了测试缺乏WlsShhCre小鼠中观察到软骨是否缺乏间质凝聚，从胎龄E12.5到E14.5气管肺组织与巴民族权力机构凝集素染色所致。在E13.5和E14.5，荧光检测为在站点间质气管软骨在那里将形成定期带。缺乏WlsShhCre胚胎的气管组织检测的荧光表明内胚Wnt信号到气管间质是必要的间充质凝聚（ 图2）。 呼吸道细胞规范气管肺组织整装免疫荧光检测Sox9的，Nkx2.1和αSMA在E11.5的表达模式。在气管组织，Sox9的表达是极限而在显影肺Sox9的在呼吸上皮细胞（ 图3A，B和C的箭头头）的周边观察编气管作为连续条的间质（ 图3A和B中的箭头）。 Nkx2.1呈现限于肺的气管上皮独特表达模式。从上皮细胞的Wnt配体的分泌防止引起Sox9的减退表达气管间质，但不影响周围型肺上皮细胞Sox9的表达。未在气管间质容易地检测αSMA的表达，但染色在喉部（黄色箭头， 图3）和胃（ 图3）的电平检测。 在发展气管细胞增殖增殖的上皮细胞与间充质气管被组织用BrdU 体内标记后检测。气管组织的切片与抗体识别的BrdU，Sox9的和αSMA染色。在气管间质增殖配置文件指示Sox9的染色的细胞进行增殖的水平比经历增殖αSMA染色的细胞的水平。这种扩散模式似乎在WlsShhCre气管（ 图4）被还原。 <img alt="图1" src="/files/ftp_upload/53910/53910fig1.jpg" /> 图1:的Wnt /β-catenin的活动是在气管间质可操作 E13.5气管肺组织，来自的Axin2-LacZ的和WlsShhCre孤立:的Axin2-LacZ的小鼠中，用X-gal（A和B）进行染色。气管肺组织切片快速红色（A&#39;，B&#39;）进行复染。注意缺少的X-gal的染色WlsShhCre;的Axin2拉克-Z小鼠没有表现出的Wnt /β-catenin的活性（B&#39;）的。从用X-gal染色LacZ基因的Axin2小鼠E14.5组织描绘未来软骨环（C）的网站;然而，的Axin2活性仅限于间充质冷凝（箭头）（D）的外周（箭头）。比例尺A，B和C = 500微米; B&#39;，D&#39;和D = 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53910/53910fig1large.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/53910/53910fig2.jpg" /> 图 2: 在显影气管组织间质缩合 E13.5气管-肺外植体与PNA凝集素染色。控制气管组织描绘地区是凝聚所示间充质细胞（箭头A </strong&gt;的信息，C）。在WlsShhCre小鼠中没有检测到间充质凝聚（B和D中的箭头）。C和D描绘在A和B所示气管-肺组织的低放大倍数。比例尺A和B = 1毫米; C和D = 2毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53910/53910fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53910/53910fig3.jpg" /> 图3:prechondrogenic整体表达模式安装气管 Nkx2.1和Sox9的染色E11.5外植体检测。面板A显示了一个控制组织的图像，而图B显示了一个20微米的对照组织的光学部分。注意缺乏WlsShhCre组织Sox9的间充质表达（在C箭头），BUSox9的外周T工作的维修（在C箭头）。在trachealmesenchyme E11.5组织的低水平检测αSMA染色，但在喉区域观察（在D和E黄色箭头），胃（S）和剩余的心脏组织（CT）（E）。 Nkx2.1在发展气管和肺部（D，E）的上皮细胞中表达。比例尺= 10微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53910/53910fig3large.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/53910/53910fig4.jpg" /> 图 4: 在prechondrogenic间质细胞的增殖图案 E13.5胚胎切片用抗BrdU的，Sox9的和αSMA抗体染色。面板C和D是A的放大倍率越高， </strong&gt;的信息和B分别。虚线表示气管上皮的限制。注意Sox9的染色增殖的细胞（箭头C）和αSMA染色增殖的细胞（箭头在D）。 T =气管，E =食道。比例尺A和B = 50微米，C和D = 20微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53910/53910fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Studying Wnt Signaling During Patterning of Conducting Airways at JoVE.com

Studying Wnt Signaling During Patterning of Conducting Airways

利用耦合到整装报告小鼠和切片染色，显微镜和体内试验方便的呼吸道的正常图案形成底层机制的分析。在这里，我们将介绍这些技术如何促进Wnt信号的气管发育过程中的分析。

研究Wnt信号传导过程中的航空公司图案

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

studying-wnt-signaling-during-patterning-of-conducting-airways

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.3K 次观看.  Cincinnati Children’s Hospital Medical Center. 利用耦合到整装报告小鼠和切片染色，显微镜和体内试验方便的呼吸道的正常图案形成底层机制的分析。在这里，我们将介绍这些技术如何促进Wnt信号的气管发育过程中的分析。 

视频: Studying Wnt Signaling During Patterning of Conducting Airways

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

利用共聚焦分析<em&gt;非洲爪蟾</em&gt;调查的Wnt和Shh形态发生素梯度的调节剂

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

科研

发育生物学

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

This video article details a straightforward in vivo methodology that can be used to systematically and efficiently characterize components of complex signaling pathways and regulatory networks in many invertebrate embryos.

简单的力量:海胆胚胎作为<em&gt;体内</em&gt;对于研究复杂的细胞与细胞间信号传导网络交互开发模式

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

小鼠胚胎的龙文化，一个系统来评价分枝的分子机制

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

生物学

Flow Cytometry and Confocal Imaging Analysis of Low Wnt Expression in Axin2-mTurquoise2 Reporter Thymocytes

Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt signaling operates via a dosage-driven mechanism, motivating the need to study and measure Wnt signaling in various cell types. Although several reporter models have been proposed to represent physiological Wnt expression, either the genetic context or the reporter protein highly influenced the validity, accuracy, and flexibility of these tools. This paper describes methods for acquiring and analyzing data obtained with the Axin2-mTurquoise2 mouse Wnt reporter model, which contains a mutated Axin2em1Fstl allele. This model facilitates the study of endogenous canonical Wnt signaling in individual cells over a wide range of Wnt activity.
This protocol describes how to fully appreciate Axin2-mTurquoise2 reporter activity using cell population analysis of the hematopoietic system, combined with cell surface markers or &#946;-catenin intracellular staining. These procedures serve as a base for implementation and reproduction in other tissues or cells of interest. By combining fluorescence-activated cell sorting and confocal imaging, distinct canonical Wnt expression levels can be visualized. The recommended measurement and analysis strategies provide quantitative data on the fluorescent expression levels for precise assessment of canonical Wnt signaling. These methods will be useful for researchers who want to use the Axin2-mTurquise2 model for canonical Wnt expression patterns.

Signaling levels are known to regulate cell fate, indicating that regulation of Wnt signaling constitutes an interesting therapeutic target. Here, we describe flow cytometry and confocal microscopy analysis methods for a robust murine canonical Wnt signaling reporter model that measures distinct Wnt signaling levels.

Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt ...

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFR&#945;). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with mesenchyme.

从诱导多能干细胞生成3D全肺类器官，用于模拟肺发育生物学和疾病

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This process requires complex and coordinated paracrine interactions between signal-sending and signal-receiving cells. Given that these interactions can occur between various types of cells from different lineages, in vivo evaluation of cell-specific defects can be challenging. The present study describes a highly reproducible method to evaluate paracrine noncanonical Wnt signaling in vitro. This protocol was designed with the ability to (1) conduct functional and molecular assessments of noncanonical Wnt signaling between any two cell types of interest; (2) dissect the role of signal-sending versus signal-receiving molecules in the noncanonical Wnt signaling pathway; and (3) perform phenotypic rescue experiments with standard molecular or pharmacologic approaches.
This protocol was used to evaluate neural crest cell (NCC)-mediated noncanonical Wnt signaling in myoblasts. The presence of NCCs is associated with an increased number of phalloidin-positive cytoplasmic filopodia and lamellipodia in myoblasts and improved myoblast migration in a wound-healing assay. The Wnt5a-ROR2 axis was identified as a crucial noncanonical Wnt signaling pathway between NCC and second heart field (SHF) cardiomyoblast progenitors. In conclusion, this is a highly tractable protocol to study paracrine noncanonical Wnt signaling mechanisms in vitro.

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

The present study outlines a highly reproducible and tractable method to study paracrine noncanonical Wnt signaling events in vitro. This protocol was applied to evaluate the impact of paracrine Wnt5a signaling in murine neural crest cells and myoblasts.

体外模拟旁非规范Wnt信号传导

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

光片荧光显微术捕捉4维图像调制剪切应力对斑马鱼心脏发育的影响

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

生物工程

禽胚胎皮肤外植体培养方案的全面回顾

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

作者聚焦:深入了解当前的实验性禽皮外植体方法

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

使用禽皮肤外植体研究组织模式和器官发生

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

研究Wnt信号传导过程中的航空公司图案

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利用共聚焦分析

简单的力量:海胆胚胎作为

小鼠胚胎的龙文化，一个系统来评价分枝的分子机制

Flow Cytometry and Confocal Imaging Analysis of Low Wnt Expression in Axin2-mTurquoise2 Reporter Thymocytes

从诱导多能干细胞生成3D全肺类器官，用于模拟肺发育生物学和疾病

体外模拟旁非规范Wnt信号传导

光片荧光显微术捕捉4维图像调制剪切应力对斑马鱼心脏发育的影响

使用禽皮肤外植体研究组织模式和器官发生

使用禽皮肤外植体研究组织模式和器官发生

使用禽皮肤外植体研究组织模式和器官发生