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。

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			<td style="width:211px;">90279</td>
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			<td height="21" style="height:21px;">4% Paraformaldehyde in PBS</td>
			<td style="width:192px;">Affymetrix</td>
			<td>19943</td>
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			<td height="21" style="height:21px;">BSA</td>
			<td style="width:192px;">Fischer Scientific&#160;</td>
			<td style="width:211px;">BP16000</td>
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			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">122262</td>
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			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">P5368-10PAK</td>
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			<td>T8787</td>
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			<td style="width:211px;">G8773</td>
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			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">T5648</td>
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			<td height="21" style="height:21px;width:381px;">Sunflower seed oil</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">S5007</td>
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			<td height="21" style="height:21px;">Tween 20</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">P1379</td>
			<td></td>
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			<td height="21" style="height:21px;width:381px;">PECAM (CD31)</td>
			<td>BD Pharmingen&#160;</td>
			<td style="width:211px;">550274</td>
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			<td>Invitrogen</td>
			<td>A-21247</td>
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			<td height="21" style="height:21px;width:381px;">DAPI nuclear stain</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">D9542</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;">37oC Incubator</td>
			<td style="width:192px;">Thermoscientific Fischer</td>
			<td style="width:211px;">Heratherm, Compact Microbiological Incubators</td>
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			<td height="21" style="height:21px;">48 well plates</td>
			<td style="width:192px;">Cell Treat</td>
			<td style="width:211px;">229148</td>
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			<td height="21" style="height:21px;width:381px;">Analytical Balance</td>
			<td style="width:192px;">Metler Toledo</td>
			<td>PB153-S/FACT</td>
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			<td height="21" style="height:21px;">Confocal microscope</td>
			<td>Zeiss</td>
			<td>Zeiss 510 confocal microscope</td>
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			<td height="21" style="height:21px;width:381px;">Disecting Microscope</td>
			<td style="width:192px;">Unitron</td>
			<td style="width:211px;">Z850</td>
			<td></td>
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			<td height="21" style="height:21px;">Fluorescent microscope</td>
			<td>Zeiss</td>
			<td>Observer. Z1</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Germinator 500 Glass Bead Sterilizer</td>
			<td style="width:192px;">CellPoint Scientific</td>
			<td style="width:211px;">GER-5287-120V</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Light Source</td>
			<td style="width:192px;">SCHOTT</td>
			<td style="width:211px;">ACE I</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Pair of Scissors</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Petri dish 60x15mm&#160;</td>
			<td align="left">TPP Techno Plastic Products AG</td>
			<td>93060</td>
			<td></td>
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			<td height="21" style="height:21px;width:381px;">Rocker II&#160; Platform Rocker</td>
			<td>Boekel Scientific</td>
			<td style="width:211px;">260350</td>
			<td></td>
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			<td height="21" style="height:21px;">Scintillating tubes</td>
			<td style="width:192px;">Fischer Scientific&#160;</td>
			<td>03-337-26</td>
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			<td height="21" style="height:21px;">Transfer pipette</td>
			<td style="width:192px;">Samco Scientific</td>
			<td style="width:211px;">202</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Tweezers</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
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</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

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

在成像钙<em&gt;果蝇</em&gt;在蛋激活

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

科研

发育生物学

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

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 ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

在细胞分化过程中光介导的可逆调节丝裂原活化蛋白激酶途径<em&gt;爪蟾</em&gt;胚胎发育

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

通过清除心脏三维成像捕获目标细胞群的心脏损伤反应

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

小鼠胚胎细胞的多路复用单细胞mRNA测序分析

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

用于微CT成像的成人和早期产后小鼠肺的血管铸造

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

成像清除胚胎和出生后心脏在单细胞分辨率

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