We thank M.W. laboratory members for scientific discussion. This work is supported by AHA [13SDG16920099] to M.W., and by National Heart, Lung, and Blood Institute grants [R01HL121700] to M.W. Images were captured in the Imaging Core Facility at the Albany Medical College.

所有动物实验的机构动物护理和使用委员会（IACUC）奥尔巴尼医学院根据NIH指南照顾和实验动物使用批准并执行。 1.解决方案的准备注:Rosa26Cre ERT2 15，R26R-16五彩纸屑 ，αMHCCre重组酶17， 和ROSA26-mTmG（mTmG）20鼠标线被购得肌钙蛋白T-18的Cre来自蛟博士在阿拉巴马大学的礼物NFATC1 Cre重组酶 。来自周斌博士医学19的爱因斯坦医学院的礼物。小鼠通过吸入二氧化碳在一个封闭的腔室安乐死为至少60秒，接着通过双边开胸，断头或颈椎脱位死亡验证的物理手段。 <ol><li>他莫昔芬准备。溶解10毫克三苯氧胺的10ml葵花籽OI的湖一旦完全溶解，等分试样并贮存在-20℃。 </li><li>制备磷酸盐缓冲盐水（PBS）中。溶解的 Na 2 HPO 4（1.41960克），KH 2 PO 4（0.24496克），氯化钠（8.0669克）和KCl（0.20129克）在1L的蒸馏水中并调节pH至7.4。 </li><li>通过在100毫升PBS中溶解100微升吐温-20的PBS中的0.1％Tween-20的溶液:准备PBST。 </li><li>准备封闭液。通过3克BSA溶解在100毫升的PBST含有0.2％Tween-20的使3％牛血清白蛋白（BSA）封闭溶液。 </li><li>准备CUBIC-1（试剂1）。混合25％（重量）的尿素，25％（重量）N，N，N&#39;，N&#39;-四（2-羟丙基）乙二胺和15％（重量）的Triton X-100在蒸馏水中2 O </li><li>准备CUBIC-2（试剂2）。混合50重量％的蔗糖，25％（重量）的尿素，10重量％2，2&#39;，2&#39;&#39; - nitrilotriethanol和0.1％（体积/体积）的Triton X-100在蒸馏水中2 O </li><li>制备的Sca 勒 A2:4M尿素，10％w / v的甘油和0.1％w / v的的Triton X-100在卫生署2 O.调节pH值至7.7。 </li><li>制备的Sca 勒 B4:8M尿素和0.1％w / v的的Triton X-100在蒸馏水中2 O调整pH值至8.7。 </li><li>制备的Sca 文件 70:4M尿素，70％w / v的甘油和0.1％（重量）用蒸馏水2 O /体积的Triton X-100 </li></ol> 2.他莫昔芬诱导，胚胎分离及固定<ol><li>他莫昔芬诱导和胚胎隔离<ol><li>使用弯曲进料管批准的协议（茶杯说教13-12007），灌胃女R26Cre ERT2五彩纸屑小鼠（R26Cre ERT2雄性小鼠插入式）与E7.75 33胚胎发育第10天微克/克三苯氧胺。在9.5天胚胎（E9.5）或E10.5，安乐死雌性小鼠用CO 2和颈脱位。 </li><li>小鼠的死亡验证后（见1注），用剪刀拆开鼠标腹腔解剖出子宫角，用剪刀和镊子，并重新的帮助去除输卵管层租赁从子宫管23,24的胚胎。 <ol><li>胚胎转移到含有PBS（pH 7.4）中的培养皿。下的解剖显微镜，删除与镊子的帮助下非胚胎层（绒毛膜，卵黄囊和羊膜）。 </li></ol></li><li>使用镊子和剪刀，收集雌性小鼠胚胎尾部样本进行基因分型如先前报道22。使用塑料移液管，每个胚胎转移到含有1ml 1×PBS中的48孔平板的孔中。 </li><li>在荧光显微镜，通过与摄像头倒置显微镜识别GFP / RFP / CFP积极的胚胎。剥离远用镊子心包和旋转具有弯曲镊子的帮助下心脏/胚，以确定荧光的克隆对心脏的两侧与GFP / RFP / CFP滤波器的数量。不需要裁员。 </li></ol></li><li>识别GFP / RFP / CFP积极的胚胎后，迅速用1X PBS洗涤胚胎两次（每次2分钟）日È48上，在室温（RT）下在板振荡器孔板。这将去除多余血液。 </li><li>修复用4％多聚甲醛（PFA）在室温胚胎/胚胎心脏。在固定时间取决于胚胎的年龄和组织的大小。为E9.5固定胚胎在室温2小时。胚胎后期胚胎需要更长的固定时间，它甚至可以延长为产后心脏24小时。 注意:多聚甲醛是有毒的，避免与皮肤，眼睛和粘膜接触。 </li><li>固定后，在48孔板洗涤胚胎/心脏两次用1×PBS（每次10分钟）上在室温在板振荡器。这从胚胎/心脏去除多余的PFA。胚胎，然后再整装染色和组织清算处理。 </li></ol> 3.整装染色注:PFA固定后胚胎/心脏如下受到整装染色。 <ol><li>通透的胚胎/心脏用1米48孔板升0.1％的PBST，在室温上板振荡器2小时。 </li><li>以下透，在4ºC浸入胚胎/心脏在1ml阻断缓冲液中，用于在板振荡器2小时或过夜。 </li><li>沉浸胚胎/心脏的内皮细胞特异性抗体PECAM（CD31），稀释（1:100），0.3毫升4ºC封闭缓冲液对板振荡器48小时。 </li><li>在板振动筛使用PBST 5次，每次30分钟在室温洗胚胎/心脏。这消除了从组织的过量的非特异性结合的一次抗体。 </li><li>浸入的Alexa Fluor 647山羊抗 - 鼠第二抗体胚/心脏，稀释（1:1,000）在1毫升，在4ºC封闭缓冲液对板振荡器48小时。 </li><li>重复步骤3.4洗二次抗体。 </li><li>后固定用4％PFA胚胎/心脏在RT上板振荡器1小时，并与每个PBS中的5分钟洗涤两次。 </li><li>与核染色4&#39;，6-二脒基-2-苯基吲哚（DAPI）（在concent在PBST中稀释染色胚胎/心脏为0.01毫克/毫升），在室温上板振荡器10分钟配给。与每5分钟2 PBS洗涤跟随。 </li></ol> 4.胚胎/心脏结算SCA的L E或三次法注:整装染色后，胚胎/心脏受到任何SCA的文件或三次组织清除的方法。方法的选择依赖于组织大小和胚胎的年龄。对于E11.5或更早年代的胚胎，用的Sca L E组织清除的方法，而对于较旧的胚胎或产后心立方组织清除方法是首选。 <ol><li>大规模组织清除方法<ol><li>在闪烁瓶中将1ml的Sca L E A 2溶液（用铝箔包裹）偶尔轻轻摇动（用手）48小时，在4℃下孵育胚胎/心脏。 </li><li>的Sca L E A2孵育后，用1毫升的Sca L E B4解决方案孵化胚胎/心脏在4℃，偶尔温和振荡ANOT同一小瓶她48小时。 </li><li>孵育胚胎/心脏再48小时用1毫升的Sca L E 70在4℃，偶尔轻轻摇动。用90％的甘油山胚（参见5.1节）。 </li></ol></li><li> CUBIC组织清除方法<ol><li>对于全胚胎/立方心脏清理，浸泡每个胚胎/心脏在1毫升CUBIC-1与偶尔的温柔在37ºC7惊天48小时。 48小时后，更换用相同体积的新鲜立方试剂1的溶液中，并在37℃下，偶尔用手摇动孵育样品另外48小时。 </li><li>在室温下用手轻轻摇动洗三次-1处理的心脏/用1×PBS几次胚胎。这消除了多余的CUBIC-1解决方案。 </li><li>用PBS洗涤三次出-1后，浸泡胚胎/心立方-2溶液（每胎/心脏1克）48小时，在37ºC与偶尔用手轻轻摇动。在此之前，胚胎/心脏使用甘油的安装（见第5节0.2）。 </li></ol></li></ol> 5.样品放置和成像注:SCA中的L E 70或立方-2解决方案清除胚胎或器官可直接与规模70和CUBIC-2溶液为成像介质分别进行成像。但是，考虑到胚胎样品的脆弱性清除试剂，如果样品不能立即成像，但长期存储，保存在90％的甘油的试样按照下面的协议。 <ol><li>直接浸泡在SCA 乐在4°C，直到成像在1毫升90％甘油和商店清除样本。 </li><li>用1×PBS洗涤三次处理的样品两次，每次5分钟，并依次孵育用1ml的30％，50％，70％和90％的甘油溶液的每个样品30分钟。 </li><li>对于成像，将样品与90％甘油和图像克隆的最小量与装有两个光子能力的共焦显微镜转移到玻璃底培养皿。 </li><li>图像中是10X / 0.3 NA甘油心脏或胚胎，一个25X / 0.8 NA浸泡，或40X / 1.2 NA水矫正物镜。图片DAPI使用2光子激发从一个连贯的钛蓝宝石激光变色龙。显微镜和物镜的选择将依赖于实验， 例如用于超分辨率的目的，一个光片的荧光显微镜可以利用。 </li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+the+epicardium+and+the+embryonic+myocardial+circulation+in+the+mouse.">The origin of the epicardium and the embryonic myocardial circulation in the mouse.</a> Anat Rec. 201, 157-168 (1981).</li><li>Wu, M., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numb+family+proteins:+novel+players+in+cardiac+morphogenesis+and+cardiac+progenitor+cell+differentiation.">Numb family proteins: novel players in cardiac morphogenesis and cardiac progenitor cell differentiation.</a> Biomolecular concepts. 6, 137-148 (2015).</li><li>Li, 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=Single-Cell+Lineage+Tracing+Reveals+that+Oriented+Cell+Division+Contributes+to+Trabecular+Morphogenesis+and+Regional+Specification.">Single-Cell Lineage Tracing Reveals that Oriented Cell Division Contributes to Trabecular Morphogenesis and Regional Specification.</a> Cell reports. 15, 158-170 (2016).</li><li>Alkaabi, K. M., Yafea, A., Ashraf, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pH+on+thermal-+and+chemical-induced+denaturation+of+GFP.">Effect of pH on thermal- and chemical-induced denaturation of GFP.</a> Appl Biochem Biotechnol. 126, 149-156 (2005).</li><li>Captur, G., 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=Morphogenesis+of+myocardial+trabeculae+in+the+mouse+embryo.">Morphogenesis of myocardial trabeculae in the mouse embryo.</a> J Anat. , (2016).</li></ol>

The authors declare no competing financial interests.

胚胎隔离是一个非常关键的步骤。 E9.5胚胎的大小很脆弱的，小的，所以格外小心，应注意不要隔离时损坏胚胎/心脏结构。非胚层外包围胚胎/心脏成像应该在整个胚胎时特别小心取出。这使得抗体和清算混合穿透深胚胎组织内部，而且在成像时去除背景信号有所帮助。包括对PECAM抗体，乙酰化α微管蛋白和β1整合多种抗体进行了检查，并全部被成功检测（数据未显示）33。这证明了分子完?...

心脏形态发生是需要的四种不同类型的心脏祖细胞的时空组织进入心脏的不同扇区的连续事件，并且还需要多基因调控网络来协调这一过程以形成功能性心脏1,2。心脏规范，分化，图案，和室成熟的心源性转录因子调控3。基因突变或这些因素在心脏祖细胞转录后像差可能导致要么胚胎致死或先天性心脏缺陷（CHD）4。心脏形态发生的研究需要在三维（3D）固有的结构细节和单标记心脏祖细胞系心脏发育过程中的跟踪将促进心脏形态的理解的理解。一些高分辨率的基础部分断层扫描方法已经在日被开发Ë过去几十年来的图像器官结构5,6;然而，这些方法需要昂贵的，专门的仪器，广泛劳动，并且缺乏在单细胞分辨率的详细结构组织在最终体积重建图像7,8。 在单细胞水平的3D体积成像提供研究祖细胞分化和细胞行为体内 7的装置。然而，组织光散射仍然是主要的障碍深完整的心脏内的3D成像的细胞和结构。脂质是光散射的一个主要来源，和去除脂质和/或脂质和它们的周围区域之间的折射率差的调整的是用于提高组织透明度8潜力的方法。在过去的几年中，一些组织结算方法的发展，从而降低组织的不透明性和光散射，像BABB（苄醇和苄benzoatË混合物）和DBE（四氢呋喃和dibenzylether）;但在这些方法中，荧光猝灭仍然是一个问题8-10。溶剂为基础的亲水性的方法，如SeeDB（果糖/硫代甘油）和3DISCO（二氯甲烷/ dibenzylether），保持荧光信号，但不使整个器官透明7,8,11。相比较而言，净度组织清除方法呈现器官透明的，但它需要一个专门的电泳装置以除去脂质8,12一样，PACT（无源清晰度技术），这也需要水凝胶包埋7,13。有关所有可用的组织清除方法的详细信息，请参阅表1 Richardson和利希特曼等。7。 2011年，哈马等人偶然发现了一种亲水的混合物"的Sca 乐 &#39;（尿素，甘油和Triton X-100混合物）呈现小鼠大脑和胚胎transpar耳鼻喉科而完全从克隆标记保持14荧光信号。这允许完整的大脑中的几个毫米和神经元群体和突起的大规模重建的深度成像以亚细胞的分辨率。须崎等人。进一步完善了Sca 乐加入氨基醇，并制定了"CUBIC"（清晰，通畅脑成像的鸡尾酒和计算分析）组织清除的方法，从而增加磷脂溶解，降低了结算时间，并允许多色荧光成像8。在本研究中，心脏发生在服用心脏内线优势在SCA 乐和立方组织清算技术和高分辨率三维光学切片，单个克隆使用Rosa26Cre ERT2 15，R26R-纸屑 16，αMHCCre重组酶17， 肌钙蛋白Cre重组酶被追踪18， </strong&gt; NFATC1 Cre重组酶 19，和ROSA26-mTmG（mTmG）20鼠标线。整个装载染色（WMS）方法的组合预先21,22与组织结算方法还允许在标记的克隆其他蛋白质的染色和对它们的行为在3D体积上下文中研究开发的。组织清除和WMS的组合允许更好地理解不同的基因和蛋白质的心脏发育过程中的作用，并先天性心脏缺陷的病因。这个协议可以被应用开发过程中来研究其他祖细胞分化，细胞行为和器官形态发生的事件。

<table><tbody><tr><td>2,2&amp;prime;,2&amp;prime;&amp;rsquo;-nitrilotriethanol&amp;nbsp;</td><td>Sigma Aldrich</td><td>90279</td><td></td></tr><tr><td>4% Paraformaldehyde in PBS</td><td>Affymetrix</td><td>19943</td><td></td></tr><tr><td>BSA</td><td>Fischer Scientific&amp;nbsp;</td><td>BP16000</td><td></td></tr><tr><td>N,N,N&amp;rsquo;,N&amp;rsquo;-tetrakis(2-hydroxypropyl) ethylenediamine&amp;nbsp;</td><td>Sigma Aldrich</td><td>122262</td><td></td></tr><tr><td>Phosphate Buffer Saline</td><td>Sigma Aldrich</td><td>P5368-10PAK</td><td></td></tr><tr><td>Triton&amp;nbsp;X-100</td><td>Sigma Aldrich</td><td>T8787</td><td></td></tr><tr><td>Urea</td><td>Sigma Aldrich</td><td>U-1250</td><td></td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>84097</td><td></td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G8773</td><td></td></tr><tr><td>Tamoxifen</td><td>Sigma Aldrich</td><td>T5648</td><td></td></tr><tr><td>Sunflower seed oil</td><td>Sigma Aldrich</td><td>S5007</td><td></td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P1379</td><td></td></tr><tr><td>PECAM (CD31)</td><td>BD Pharmingen&amp;nbsp;</td><td>550274</td><td></td></tr><tr><td>Alexa Fluor 647&amp;nbsp;</td><td>Invitrogen</td><td>A-21247</td><td></td></tr><tr><td>DAPI nuclear stain</td><td>Sigma Aldrich</td><td>D9542</td><td></td></tr><tr><td>37&lt;sup&gt;o&lt;/sup&gt;C Incubator</td><td>Thermoscientific Fischer</td><td>Heratherm, Compact Microbiological Incubators</td><td></td></tr><tr><td>48 well plates</td><td>Cell Treat</td><td>229148</td><td></td></tr><tr><td>Analytical Balance</td><td>Metler Toledo</td><td>PB153-S/FACT</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>Zeiss 510 confocal microscope</td><td></td></tr><tr><td>Disecting Microscope</td><td>Unitron</td><td>Z850</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Zeiss</td><td>Observer. Z1</td><td></td></tr><tr><td>Germinator 500 Glass Bead Sterilizer</td><td>CellPoint Scientific</td><td>GER-5287-120V</td><td></td></tr><tr><td>Light Source</td><td>SCHOTT</td><td>ACE I</td><td></td></tr><tr><td>Pair of Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Petri dish 60 mm x15 mm&amp;nbsp;</td><td>TPP Techno Plastic Products AG</td><td>93060</td><td></td></tr><tr><td>Rocker II&amp;nbsp; Platform Rocker</td><td>Boekel Scientific</td><td>260350</td><td></td></tr><tr><td>Scintillating tubes</td><td>Fischer Scientific&amp;nbsp;</td><td>03-337-26</td><td></td></tr><tr><td>Transfer pipette</td><td>Samco Scientific</td><td>202</td><td></td></tr><tr><td>Tweezers</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr></tbody></table>

imaging cleared embryonic and postnatal hearts at single-cell resolution

在全心脏水平单克隆跟踪和分析可以心脏发育过程中确定心脏祖细胞的行为和分化，并允许正常和异常的心脏形态发生的细胞和分子基础的研究。回顾单克隆分析最近的新兴技术使心脏形态发生在单细胞分辨率的研究是可行的。然而，组织的不透明度和心脏的成像深度的光散射在单细胞分辨率提高阻碍全心脏成像。为了克服这些障碍，整个胚胎清算系统，可以使心脏的高度透明为照明和检测必须发展。幸运的是，在过去的几年中，已报告的全生物结算系统如透明度，爱生雅乐 ，SeeDB，ClearT，3DISCO，CUBIC，DBE，BABB和PACT许多方法。这个实验有兴趣卡的细胞和分子机制IAC形态。心脏发育过程中ROSA26-纸屑系统稀疏标签细胞;最近，我们建立了单细胞系通过ROSA26 Cre重组ERT2跟踪。我们适应几个全胚胎清算方法，包括了Sca LE和立方（清晰，通畅脑成像的鸡尾酒和计算分析）来清除组合与胚胎整装染色心脏内部的图像单一克隆。该心脏在单细胞分辨率被成功成像。我们发现的Sca 乐可以清除胚胎心脏，但不能有效地清除产后心脏，而CUBIC可以清除产后心脏，但损害胚胎心脏的组织溶解。此处所描述的方法将允许基因功能的研究在心脏形态发生，这反过来，可以揭示先天性心脏缺陷的细胞和分子基础中的单个克隆的分辨率。

成像清除胚胎心脏脊椎动物心脏的形成是一个spatiotemporally调节形态发生过程，并且取决于从四个不同来源1的组织和祖细胞的分化。从心脏月牙的第一场心脏细胞会向着腹中线折叠，形成线性心脏管。来自第二心脏视野中的细胞，最初驻留dorsomedially到第一心脏字段，随后转位到咽和内脏中胚层，从那里它们迁移到线性?...

Watch this Scientific Journal Video about Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution at JoVE.com

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

成像清除胚胎和出生后心脏在单细胞分辨率

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

imaging-cleared-embryonic-postnatal-hearts-at-single-cell

Research

JoVE Journal

Developmental Biology

8.1K 次观看.  Albany Medical College. We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately. 

视频: Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

在胚胎小鼠心脏采用免疫荧光心肌发展分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

科研

发育生物学

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

在清除胚胎心脏冠状血管分析

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

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

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

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

基于微注射的免疫胚胎心肌细胞体内植入系统

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

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

小鼠胚胎中早期心脏祖细胞的实时成像

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

免疫染色的解剖斑马鱼胚胎心脏

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

生物学

用于全面心脏成像的光片显微镜和组织透明化

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights into fundamental cardiac physiology and pathologic responses. We hereby delve into the development and implementation of the LSM technique to elucidate the micro-architecture of the heart in mouse models. The methodology integrates a customized LSM system with tissue clearing techniques, mitigating light scattering within cardiac tissues for volumetric imaging. The combination of conventional LSM with image stitching and multiview deconvolution approaches allows for the capture of the entire heart. To address the inherent trade-off between axial resolution and field of view (FOV), we further introduce an axially swept light-sheet microscopy (ASLM) method to minimize out-of-focus light and uniformly illuminate the heart across the propagation direction. In the meanwhile, tissue clearing methods such as iDISCO enhance light penetration, facilitating the visualization of deep structures and ensuring a comprehensive examination of the myocardium throughout the entire heart. The combination of the proposed LSM and tissue clearing methods presents a promising platform for researchers in resolving cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

作者聚焦：用于研究啮齿动物心脏心肌结构的定制光片成像

The protocol utilizes advanced light-sheet microscopy along with adapted tissue clearing methods to investigate intricate cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

光片成像揭示啮齿动物心脏的心脏结构

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights ...

生物工程

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

细胞标记和注射胚胎发展小鼠心脏

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

成像清除胚胎和出生后心脏在单细胞分辨率

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在胚胎小鼠心脏采用免疫荧光心肌发展分析

在清除胚胎心脏冠状血管分析

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

基于微注射的免疫胚胎心肌细胞体内植入系统

小鼠胚胎中早期心脏祖细胞的实时成像

免疫染色的解剖斑马鱼胚胎心脏

光片成像揭示啮齿动物心脏的心脏结构

光片成像揭示啮齿动物心脏的心脏结构

光片成像揭示啮齿动物心脏的心脏结构

细胞标记和注射胚胎发展小鼠心脏