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>

<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. M., Fox, P. L., Mjaatvedt, C. H., Hoffman, S., Capehart, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+method+for+analysis+of+chondrogenesis+in+limb+mesenchyme+from+individual+transgenic+(hdf)+embryos.">An in vitro method for analysis of chondrogenesis in limb mesenchyme from individual transgenic (hdf) embryos.</a> Methods Cell Sci. 25, 97-104 (2003).</li><li>Cohen, E. D., 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=Wnt+signaling+regulates+smooth+muscle+precursor+development+in+the+mouse+lung+via+a+tenascin+C/PDGFR+pathway.">Wnt signaling regulates smooth muscle precursor development in the mouse lung via a tenascin C/PDGFR pathway.</a> J Clin Invest. 119, 2538-2549 (2009).</li><li>Boucherat, O., 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=Partial+functional+redundancy+between+Hoxa5+and+Hoxb5+paralog+genes+during+lung+morphogenesis.">Partial functional redundancy between Hoxa5 and Hoxb5 paralog genes during lung morphogenesis.</a> Am J Physiol Lung Cell Mol Physiol. 304, L817-L830 (2013).</li></ol>

"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<em...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>K3Fe(CN)6</td>
			<td>Sigma</td>
			<td>P8131</td>
			<td></td>
		</tr>
		<tr>
			<td>K4Fe(CN)6</td>
			<td>Sigma-Aldrich</td>
			<td>P3289</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</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>NP4O</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&#160;&#160;&#160; ( 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&#160; 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.&#160;</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?...

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

Universidad de Cartagena UDEC

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

Method of Studying Palatal Fusion using Static Organ Culture

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that adheres to form the midline epithelial seam (MES). The theories suggest that MES cells follow an epithelial to mesenchymal transition (EMT), apoptosis and migration, making a fused palate 1. Complete disintegration of the MES is the final essential phase of palatal confluence with surrounding mesenchymal cells. We provide a method for palate organ culture. The developed in vitro protocol allows the study of the biological and molecular processes during fusion. The applications of this technique are numerous, including evaluating responses to exogenous chemical agents, effects of regulatory and growth factors and specific proteins. Palatal organ culture has a number of advantages including manipulation at different stages of development that is not possible using in vivo studies.

Studies of palate development are motivated by the incidence of cleft palate, a birth defect that imposes a tremendous health burden and can leave lasting disfigurement. We demonstrate here a technique to culture palatal shelves that can be used to study different signaling pathways involved in palatal development and fusion.

使用静态器官培养研究腭融合的方法

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

科研

发育生物学

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 ...

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 ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

在鼠标破骨细胞前体Notch信号的刺激

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid fissure closure. Recently, the identification of individuals with coloboma in the superior aspect of the iris, retina, and lens led to the discovery of a novel structure, referred to as the superior fissure or superior ocular sulcus (SOS), that is transiently present on the dorsal aspect of the optic cup during vertebrate eye development. Although this structure is conserved across mice, chick, fish, and newt, our current understanding of the SOS is limited. In order to elucidate factors that contribute to its formation and closure, it is imperative to be able to observe it and identify abnormalities, such as delay in the closure of the SOS. Here, we set out to create a standardized series of protocols that can be used to efficiently visualize the SOS by combining widely available microscopy techniques with common molecular biology techniques such as immunofluorescent staining and mRNA overexpression. While this set of protocols focuses on the ability to observe SOS closure delay, it is adaptable to the experimenter&#39;s needs and can be easily modified. Overall, we hope to create an approachable method through which our understanding of the SOS can be advanced to expand the current knowledge of vertebrate eye development.

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Here, we present a standardized series of protocols to observe the superior ocular sulcus, a recently-identified, evolutionarily-conserved structure in the vertebrate eye. Using zebrafish larvae, we demonstrate techniques necessary to identify factors that contribute to the formation and closure of the superior ocular sulcus.

达尼奥胚胎发生过程中的上眼硫可视化

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid ...

Genotyping of Sea Anemone during Early Development

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the life of the animal. Following in vitro fertilization and de-jellying, zygotes are allowed to develop for 24 h at room temperature to reach the early- to mid-gastrula stage. The gastrula embryos are then placed on an agarose gel bed in a Petri dish containing seawater. Under the dissecting microscope, a tungsten needle is used to surgically separate an aboral tissue fragment from each embryo. Post-surgery embryos are then allowed to heal and continue development. Genomic DNA is extracted from the isolated tissue fragment and used as a template for locus-specific PCR. The genotype can be determined based on the size of PCR products or presence/absence of allele-specific PCR products. Post-surgery embryos are then sorted according to the genotype. The duration of the entire genotyping process depends on the number of embryos to be screened, but it minimally requires 4&#8211;5 h. This method can be used to identify knockout mutants from a genetically heterogeneous population of embryos and enables analyses of phenotypes during development.

The goal of this protocol is to genotype the sea anemone Nematostella vectensis during gastrulation without sacrificing the embryo.

早期开发期间海葵基因分型

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

毛因神经发生期间诺奇信号动力学的实时生物发光成像

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

研究Wnt信号传导过程中的航空公司图案

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