The authors would like to thank Dr. George Gittes for advice and expertise throughout this study. SSL was supported by an American Heart Association fellowship (11POST7330002). Further to this SSL and this study was supported by an NIDDK Mentored Research Scientist Development Award (DK096996) and by the Children&#8217;s Hospital of Pittsburgh.

注：匹兹堡机构动物护理和使用委员会的批准大学的所有实验。 1.制备超声仪器显微注射和胚胎<ol><li>建立阶段，安装，和探针（ 图1）以及外科手术器械（ 图2）。地方的磷酸盐缓冲盐水（PBS）溶液（pH7.4）的在37℃的暖浴。完全填充微注射针与矿物油，用1ml注射器附连到第二柔性地下25针，通过其基极。 </li><li>固定针到旋转支架臂上，与矿物油溶液空针。重新填充2.5微升的TL解决方案。确保没有气泡注射针中存在。旋转针手臂朝着墙，离舞台。 </li><li>通过异氟醚持续输注麻醉孕妈妈在麻醉室。当妈妈呈现昏迷，麻醉转移到鼻管放置在C舞台audal侧，并与炉鼻的鼻管仰卧位的地方母亲以允许延续的完全麻醉状态。 </li><li>磁带肢体45°角，或手和脚休息和放置上层建筑ECG /温度显示器标签。重要的是，在怀孕的坝被连续监测，以确保麻醉足以并向眼睛软膏施加到降低干燥。 </li><li>申请脱毛产品横跨母亲的下腹部，轻轻地用干棉签擦去，然后再次用70％的乙醇饱和棉签除去任何过量的产物和头发。确保现场切口（ 图3）清洁腹部皮肤关闭所有的头发。 </li><li>执行对孕妈妈用细手术镊子和剪刀剖腹探查术。请注意，以避免削减任何重大的血管或脏器。使第一，表皮直切口1.5-2厘米以上的阴道和继续削减对肋骨的approximat伊利2-3厘米。露出皮下膜，找到白线（“白线”）（ 图3），并切平行于初始切口，沿着相同的长度，以露出内部的内脏器官和子宫saccules。 </li></ol> 2.提取胚胎<ol><li>使用6英寸棉签涂抹机动母亲和胚胎。轻轻推动（不要用力）对皮肤敷贴操纵第一胎出切口开放。提取第一胚胎球囊后，轻轻地，慢慢地，并到母亲的外部拉子宫角的其余部分。避免因切口在这个阶段拉肠或其他器官。 </li><li>量化胚胎和轻轻地放在左宫角回妈妈。然后开始把右侧子宫角回母亲开始从喇叭阴道胚胎球囊最远和向下移动。继续，直到只有两个胚胎仍然暴露（ 网络连接古尔4）。最后，在上述的柱，并平行的位置胚胎切口线，作为识别不切断子宫循环。 </li><li>放置粘土块和位置手臂和身体，以及腿和尾部之间。确保粘土表面大约级别与开腹手术切口。湿窗孔的培养皿37℃PBS。 </li><li>把握延伸钳子用右手和有孔培养皿左手（平行于胚胎开窗），并通过开窗术带来封闭镊子〜5厘米，从培养皿的顶部。打开钳完全没有撕裂开窗网。 </li><li>操纵手以上的胚胎和视线集中在这两个胚胎saccules。如果没有（或轻度）触摸saccules与开窗啮合或镊子，透过缝隙滑到他们并设置培养皿到母亲的腹部。用钳子还在延续，操作有孔的网状座位它放在一边，并在每个胚胎的基地（ 图5 </st荣&gt;）和拉镊子从切口开放。 </li><li>下一步，将装有墙培养皿中的蓝色橡胶，而不会挤压或伤害胚胎（ 图6）。确保黏土块坚决平衡培养皿中，胚胎，以及含有橡胶墙。最后，填充培养皿用37℃的PBS中直至胚完全淹没（ 图6），同时控制用于 ​​在有孔啮合泄漏。 </li></ol> 3.注射程序<ol><li>较低的注射阶段与母亲和胚胎向下使用的Z-电平调整旋钮（ 图1），然后旋转注射针及安装臂，并直接与超声波探头排队，以针半厘米的左侧和下探针尖端（ 图7） 。推针臂（未针）45°远离探头。要小心，不要打了菜或超声探头的针尖。 </li><li>提高注射阶段回到原来的高度，用超声波P长袍的正上方胚胎探头必须稍微淹没，并在3-4毫米的胚胎组织。利用X＆Y阶段调节旋钮（ 图8），修订胚/妈妈/舞台上的地位，有系统地找到跳动胚胎心脏。 </li><li>直接超声波屏幕的中心观察到黑中心目标标记绘制（*）。找到左心室（最好）或中庭采用X＆Y级调节旋钮（ 图8），然后抬高或使用旋转旋钮，在舞台上通过超声波屏幕上的黑中心标记精确定位注入目标降低的阶段。 </li><li>使用X级调节旋钮，将胚胎到右侧，关闭超声波屏。重新定位显微注射针和臂回原位，半厘米的距离和90°的超声探头（ 图7）。 </li><li>用显微注射装入调节旋钮（ 图9C-G）的位置的针与尖端清楚人igned与中心标记。调整喷射角（使用旋钮在图9C和EG），并且X，Y，和微针的Z-载体，以确保针尖被聚焦在正确的平面。仅仅使用了“注入”旋钮（ 图9F）收回显微注射针，注意不要调整其他尺寸。 </li><li>同样，使用单独的X微调阶段旋钮招回胚胎探头下，用目标正好在超声屏幕上的中心标志。确保左心室现在正好在相同的X，Y和Z平面。 </li><li>接着，只用“注入”的显微注射调节器架，慢慢穿刺胚胎与显微注射针和逐渐使前端进入左心室。注满2.5微升TL溶液进入左心室或直到空的警告提示音，通过举办“空”，挖掘“补”一次（ 图2A和B）。此时注射观察影子从它进入心脏腔室中的TL（ 图10）的针的尖端发出。相反，快速收回注射针旋转的显微注射的“注入”按钮（ 图9F）安装在注射完成。 </li><li>继续到下一个胚胎，然后重复此步骤如下这一系列的步骤剩余的胚胎，最后将胚胎最终回大坝的腹部。改用麻醉室中，轻轻移动的母亲在这里15分钟从最后一次注射时间。 </li></ol> 4.收获胚胎及分析<ol><li>通过颈椎脱位牺牲大坝。展开开腹手术切口容易地从母亲子宫中删除角。保持内子宫囊的胚胎。将在PBS冷冻胚胎。 </li><li>从解剖子宫的胚胎囊（如果进行基因分型需要收集组织），并把整个胚胎我ñ4％多聚甲醛（PFA）过夜，然后到30％蔗糖过夜，冻结在低温恒温器切片中。然后切冷冻切片，并将其放置到幻灯片免疫组化分析。任选地，使用的组织为通过脱水在100％的甲醇，在4％PFA以下定影整个安装。 </li><li>免疫组化分析，将进入冰冻切片PBS去除十月然后方框，用10％正常驴血清的部分30分钟。然后添加PECAM一级抗体以1：100稀释过夜，在4℃。第二天，洗涤载玻片中的PBT 3次，每次10分钟，并添加驴抗鼠594仲和孵育1小时，在室温。 注：这是一个非常苛刻的技术，需要超和显微注射的优化和协调。有一个非常陡峭的学习曲线，与此相关的技术，需要四到六周注射辅导就会有一个精通之前。 </li></ol>

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	<li>Abrahamson, D. R., Robert, B., Hyink, D. P., St John, P. L., Daniel, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+and+formation+of+microvasculature+in+the+developing+kidney.">Origins and formation of microvasculature in the developing kidney.</a> Kidney international. Supplement. 67, S7-S11 (1998).</li><li>Sims-Lucas, S., 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=Endothelial+Progenitors+Exist+within+the+Kidney+and+Lung+Mesenchyme.">Endothelial Progenitors Exist within the Kidney and Lung Mesenchyme.</a> PloS one. 8 (6), e65993 (2013).</li><li>Dressler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+early+kidney+specification,+development+and+patterning.">Advances in early kidney specification, development and patterning.</a> Development. 136 (23), 3863-3874 (2009).</li><li>Costantini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+controls+and+cellular+behaviors+in+branching+morphogenesis+of+the+renal+collecting+system.">Genetic controls and cellular behaviors in branching morphogenesis of the renal collecting system.</a> Wiley Interdiscip Rev Dev Biol. 1 (5), 693-713 (2012).</li><li>Costantini, F., Kopan , R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+a+complex+organ:+branching+morphogenesis+and+nephron+segmentation+in+kidney+development.">Patterning a complex organ: branching morphogenesis and nephron segmentation in kidney development.</a> Dev Cell. 18 (5), 698-712 (2010).</li><li>Das, A., 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=Stromal-epithelial+crosstalk+regulates+kidney+progenitor+cell+differentiation.">Stromal-epithelial crosstalk regulates kidney progenitor cell differentiation.</a> Nat Cell Biol. 15 (9), 1035-1044 (2013).</li><li>Rymer, C., 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=Renal+blood+flow+and+oxygenation+drive+nephron+progenitor+differentiation.">Renal blood flow and oxygenation drive nephron progenitor differentiation.</a> Am J Physiol Renal Physiol. , (2014).</li><li>Shah, S. R., 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=Embryonic+mouse+blood+flow+and+oxygen+correlate+with+early+pancreatic+differentiation.">Embryonic mouse blood flow and oxygen correlate with early pancreatic differentiation.</a> Developmental biology. 349 (2), 342-349 (2011).</li><li>Andres, A. C., 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=EphB4+receptor+tyrosine+kinase+transgenic+mice+develop+glomerulopathies+reminiscent+of+aglomerular+vascular+shunts.">EphB4 receptor tyrosine kinase transgenic mice develop glomerulopathies reminiscent of aglomerular vascular shunts.</a> Mech Dev. 120 (4), 511-516 (2003).</li><li>Wagner, R., 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=High-resolution+imaging+of+kidney+vascular+corrosion+casts+with+Nano-CT.">High-resolution imaging of kidney vascular corrosion casts with Nano-CT.</a> Microsc Microanal. 17 (2), 215-219 (2011).</li></ol>

The authors declare that they have no competing financial interests.

注射麻醉和时间框架与关于母亲的麻醉，这是至关重要的，以保持气流不变（2-3升/分钟），并在低的PSI。镇静药流必须在约1.75-2升/分举行。同时，时间安排在注射发生必须密切监测和控制与每个垃圾。对于每一个垃圾注射过程应保持在45分。这一时限是极为重要的实验，因为每个胚胎必须保持恒定，正常的心脏速率的范围内，以促进在整个身体和感兴趣器官受控和恒定的灌?...

在胚胎发育两个分立的，但同时，血管过程发生：血管生成，该过程从一个主要的预先存在的血管，和血管生成，这是一种新形成血管的来自住宅内皮祖1,2-由此容器增长。分别，前者是同义的血流量，而后者被认为主要是发生在没有它。 同时，以血管形成，肾脏祖细胞的合成，增殖和分化周期性和动态的过程开始展开上9.5天胚胎（E9.5）。在这一点上输尿管芽（UB）侵入背侧到包围后肾间质（MM），并继续直到出生3。反复分支UB进入快速冷凝后肾间质帽开始肾脏的功能单位，肾的形成。随着每一代新UB和NEPH的罗恩，老一辈流离失所到内皮层和髓质的地区，在那里他们再进行内主要血管密集的环境中进一步成熟和分化。就证明了德雷斯勒等人 3，此胚胎过程是由感应信令沉淀，如UB和MM，和细胞外因素3-6无数之间的串扰。最近的两个研究发展胰腺内的细胞外因素和肾脏包括氧张力和血流量7,8。后者将在更详细地相对于肾脏发育中讨论如下。 以暴露所述感应作用血流可能起着肾祖细胞的分化，以及在其他器官的过程，胚胎血流映射的精确和准确的方法是必要的。 计量血流的替代方法包括微升的处方trasound成像及树脂注塑9,10。决定性，这些模式已经被证明是固有地缺乏其能力，以同时地揭开血流之间的时间和空间的并置和干细胞的分化。树脂管型，例如，提供成人组织中血管形成图案的一个有效的模型，然而，在未成熟的血管，如与胚胎的时间点，容器是非常不发达和漏。因此，树脂注塑未能微小的，常常多孔，船内举行。 对于这些明显的障碍，除其他外，我们选择了把超声引导体内心内胚胎番茄凝集素（TL）显微注射到我们的肾发展的调查工作。在此过程中，我们利用超声波探头向同步引导装入微量针填充有2.5微升的TL溶液注入小鼠胚胎在E11.5，E13.5，E15.5和E17.5的时间点左心室。 E170.5是最新的发育年龄为针是没有强大到足以穿透更发达的胚胎。 这种显微注射方法的优点是丰富的。超声引导下注射允许注射针的胚胎左心室内精确定位，被动和控制驱逐溶液进入动物，对心脏的损害最小和周围组织的心脏跳动，并避免突发性心脏衰竭而死亡胚胎前全身灌注。与使用FITC标记TL的，任何灌注脉管将保持沿着其内皮顶膜的标志物。与免疫相结合，利用粘附分子（CD31，血小板内皮细胞粘附分子）和其他各种血管标记，我们能够清楚地灌注和未灌注血管区分，以及表征周围组织中的任何异常染色。

<table border="0" cellpadding="0" cellspacing="0" width="981">
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Name of Material/ Equipment</td>
			<td style="width:292px;">Company</td>
			<td style="width:164px;">Catalog Number</td>
			<td style="width:225px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Sigma Aldrich</td>
			<td>022M4004V</td>
			<td>concentration 1:5000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pecam</td>
			<td>BD Biosciences</td>
			<td>553370</td>
			<td>concentration 1:100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FITC-Tomato Lectin</td>
			<td>Vector Laboratories</td>
			<td>FL-1321</td>
			<td>concentration 2.5&#181;L / embryo</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa Fluor-594 (Donkey Anti-Rat )</td>
			<td>Jackson Immunoresearch</td>
			<td>712-585-150</td>
			<td>concentration 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microinjection Needle</td>
			<td>Origio Mid Atlantic Devices</td>
			<td>C060609</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mineral Oil</td>
			<td style="width:292px;">Fisher Scientific</td>
			<td style="width:164px;">BP26291</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mL syringe</td>
			<td style="width:292px;">Fisher Scientific</td>
			<td style="width:164px;">03-377-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Clay Blocks</td>
			<td style="width:292px;">Fisher Scientific</td>
			<td style="width:164px;">HR4-326</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Tape</td>
			<td style="width:292px;">Fisher Scientific</td>
			<td style="width:164px;">18-999-380</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td style="width:292px;">Fisher Scientific</td>
			<td style="width:164px;">NC9763655</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hair Removal Product</td>
			<td style="width:292px;">Fisher Scientific</td>
			<td style="width:164px;">NC0132811</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Scissors</td>
			<td style="width:292px;">Fine Science tools</td>
			<td style="width:164px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine Forceps</td>
			<td style="width:292px;">Fine Science tools</td>
			<td style="width:164px;">11064-07</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Surgical Marking Pen</td>
			<td style="width:292px;">Fine Science tools</td>
			<td style="width:164px;">18000-30</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Right angle forceps (for hysterectomy)</td>
			<td style="width:292px;">Fine Science tools</td>
			<td style="width:164px;">11151-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

in utero intra-cardiac tomato-lectin injections on mouse embryos to gauge renal blood flow

形成和发展肾脏血管（除了肾小球）灌注大大充分研究。作为脉管经由血管生成的发展（其为分支大血管的）和血管（ 从头血管形成），如树脂管型， 在体内超声成像，和显微解剖灌注映射技术在证明之间的亲密关系被限制胚胎内这两个过程和显影肾结构。在这里，我们描述了在小鼠胚胎在子宫内的心内超声引导FITC标记番茄凝集素显微注射的方法，以评估肾灌注的个体发育。番茄凝集素（TL）被灌注在整个胚胎和肾脏收获。组织中共同染色关于各种肾结构，包括：肾祖细胞，肾单位的结构，输尿管上皮细胞，和血管。起始于E13.5大口径血管灌流，但是周边船只仍unperfused。由E15.5和E17.5，小末梢血管以及肾小球开始变得灌注。这个实验技术对于胚胎发育过程中学习脉管和血流的作用是至关重要的。

血管的形成先于肾脏发育流大多数胚胎组织（包括肾脏）中包含的致密脉管（包括unperfused和灌注），即使是在早期胚胎的时间点。到肾脏发育中更好地衡量和分析血流我们利用在宫内胚胎心内显微注射的方法。与使用一个高分辨率超声在E11.5通过E17.5识别胚胎心脏，并按照通过剖腹提取和单一子宫球囊的曝光，我们能够注入2.5微升TL的（异硫氰酸荧光素素（FITC）缀合）。 <p...

Watch this Scientific Journal Video about In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow at JoVE.com

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

在对小鼠胚胎子宫内心脏番茄凝集素注射到计肾血流量

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis ...

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Research

JoVE Journal

Developmental Biology

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

9.6K 次观看.  Children's Hospital of Pittsburgh of UPMC. This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

视频: In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

对比成像的小鼠胚胎使用高频超声

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

科研

发育生物学

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

小鼠海马组织的前子宫内电和器官切片文化

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

在小鼠胚胎干细胞结构功能研究使用重组酶介导的盒式交换

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

小鼠血睾丸屏障完整性的一种活体研究方法

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

使用流动细胞测量来检测和量化正在发育的斑马鱼中改变的血液形成

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

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 ...

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.