Name: analysis of cardiac chamber development during mouse embryogenesis using whole mount epifluorescence
Uploaded: 2019-04-17T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence at JoVE.com

这项工作得到了 NIH R03 HL140264 (Y.J) 的支持。N) 和吉列德科学研究学者项目 (y.-j。N)。

所有动物手术都是在范德比尔特大学医疗中心机构动物护理和使用委员会的批准下进行的。
1. 小鼠胚胎的采集和解剖
<ol><li>将8-10 大的女性 Mlc-2v-tto薯 +/-与8-10 龄的男性 Mlc-2v-tto薯+/-小鼠,以获得 MLC-2v-tdTomato+/+, Mlc-2v-tttocoto+/-和 mlc-2v-tttocoto-/---胚胎。</li>
	<li>每天早上检查水坝是否有阴道塞。阴道塞检测当天中午被认为是 E0.5。 注: 阴...

<ol>
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P., 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=Atrial+identity+is+determined+by+a+COUP-TFII+regulatory+network.">Atrial identity is determined by a COUP-TFII regulatory network.</a> Developmental Cell. 25 (4), 417-426 (2013).</li><li>Nelson, D. O., Jin, D. X., Downs, K. M., Kamp, T. J., Lyons, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irx4+identifies+a+chamber-specific+cell+population+that+contributes+to+ventricular+myocardium+development.">Irx4 identifies a chamber-specific cell population that contributes to ventricular myocardium development.</a> Developmental Dynamics. 243 (3), 381-392 (2014).</li><li>Bao, Z. Z., Bruneau, B. G., Seidman, J. G., Seidman, C. E., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+chamber-specific+gene+expression+in+the+developing+heart+by+Irx4.">Regulation of chamber-specific gene expression in the developing heart by Irx4.</a> Science. 283 (5405), 1161-1164 (1999).</li><li>Kubalak, S. W., Miller-Hance, W. C., O'Brien, T. X., Dyson, E., Chien, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chamber+specification+of+atrial+myosin+light+chain-2+expression+precedes+septation+during+murine+cardiogenesis.">Chamber specification of atrial myosin light chain-2 expression precedes septation during murine cardiogenesis.</a> Journal of Biological Chemistry. 269 (24), 16961-16970 (1994).</li><li>Bardot, P., 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=The+TAF10-containing+TFIID+and+SAGA+transcriptional+complexes+are+dispensable+for+early+somitogenesis+in+the+mouse+embryo.">The TAF10-containing TFIID and SAGA transcriptional complexes are dispensable for early somitogenesis in the mouse embryo.</a> Development. 144 (20), 3808-3818 (2017).</li><li>Zhang, Z., Nam, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+MLC-2v-tdTomato+knock-in+reporter+mouse+line.">Generation of MLC-2v-tdTomato knock-in reporter mouse line.</a> Genesis. , (2018).</li><li>Ieda, M., 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=Direct+reprogramming+of+fibroblasts+into+functional+cardiomyocytes+by+defined+factors.">Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.</a> Cell. 142 (3), 375-386 (2010).</li><li>Liang, X., 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=HCN4+dynamically+marks+the+first+heart+field+and+conduction+system+precursors.">HCN4 dynamically marks the first heart field and conduction system precursors.</a> Circulation Research. 113 (4), 399-407 (2013).</li><li>Nam, Y. 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=Induction+of+diverse+cardiac+cell+types+by+reprogramming+fibroblasts+with+cardiac+transcription+factors.">Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors.</a> Development. 141 (22), 4267-4278 (2014).</li><li>Spater, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+HCN4++cardiomyogenic+progenitor+derived+from+the+first+heart+field+and+human+pluripotent+stem+cells.">A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells.</a> Nature Cell Biology. 15 (9), 1098-1106 (2013).</li><li>O'Brien, T. X., Lee, K. J., Chien, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positional+specification+of+ventricular+myosin+light+chain+2+expression+in+the+primitive+murine+heart+tube.">Positional specification of ventricular myosin light chain 2 expression in the primitive murine heart tube.</a> Proceedings of the National Academy of Sciences of the United States of America. 90 (11), 5157-5161 (1993).</li></ol>

这里描述的方法是相对简单的检查心室室的发展, 而不进行劳动密集型实验, 以标记心室或心脏特异性结构基因或蛋白质。因此, 这种方法最大限度地减少了通常在免疫抑制的心脏部分发现的技术变异性。
成功实施这种方法有两个关键步骤, 包括精确估计小鼠的胚胎年龄和胚胎心脏的解剖。我们实际上是通过识别雌性小鼠的阴道插头来估计小鼠的胚胎年龄。然而, 阴道堵塞的存?...

心脏发育过程中的腔形成是一个复杂的过程,通过几个形态上不同的胚胎阶段 1,2进行过渡。心脏祖细胞的新月形态形成一个线性心脏管, 然后经过伸长和循环, 形成发育中的心脏的螺旋状。在它的败血症过程后, 发育中的心脏转化为四腔的心脏。这些过程中的任何一个都会导致发育性心脏缺陷。因此, 了解心脏发育过程中室形成的分子机制是很重要的。尽管以前对心脏发育进行了许多研究, 但我们对这一复杂过程的理解仍然有限。
原位杂交、免疫组织化学和β-半乳糖苷酶染色使用 lacz 记者小鼠已被广泛用于研究小鼠心脏发育过程中的腔体形成, 通过标记心脏特异性或腔体特异性结构基因或蛋白质 (例如, nppa、coup-tfii、irx4、mlc-2a 和 mlc-2v)3、4、5、6、7、8、9、10.然而, 这些使用小鼠胚胎的实验需要大量的时间和专业知识, 因为必须按顺序执行几个不同的实验步骤11。在这里, 我们描述了一个简单的整体安装上皮荧光显微镜方法, 以可视化发展中的心室使用胚胎解剖从 Mlc-2v-t分解番茄记者敲门砖 12.与以前使用的方法相比, 这种方法的优点是避免复杂的实验步骤, 这些步骤往往会造成实验变化。该方案的主要目的是描述如何解剖小鼠胚胎和发展心脏, 并检查每个阶段的小鼠心脏室发展, 而无需繁琐的组织化学实验。这种方法可以很容易地应用于评估心脏发育使用各种其他转基因小鼠线标记早期心脏标记 (例如, Mesp1cor:roas26eyfp13, isl1cor:roasa26eyfp13, hcn4h2bgfp14, Hcn4cre: rosa mt"mgg14, Nkx2-5core:rosa mtmg14, Hcn4-egfp15, isl1cor:rosa Mt/2 g 14, Nkx2.5 Cre:roas26ttomato15和 tgmef2c ahf-gfp16小鼠).

<table border="0" cellpadding="0" cellspacing="0" style="width:647px;" width="647">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">dissecting microscope</td>
			<td style="width:83px;">Leica</td>
			<td style="width:119px;">MZ125</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DNA ladder (100 bp)</td>
			<td style="width:83px;">Promega</td>
			<td style="width:119px;">G2101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">epifluorescence dissecting microscope</td>
			<td style="width:83px;">Leica</td>
			<td style="width:119px;">M165 FC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">GoTaq Green master Mix</td>
			<td style="width:83px;">Promega</td>
			<td style="width:119px;">M712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">PCR machine (master cycler)</td>
			<td style="width:83px;">Eppendorf</td>
			<td style="width:119px;">6336000023</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of cardiac chamber development during mouse embryogenesis using whole mount epifluorescence

该协议的目的是描述一种方法, 解剖小鼠胚胎和可视化胚胎小鼠室在心脏发育过程中使用心室特异性荧光记者敲入小鼠 (Mlc-2v-tdtocoto 小鼠)。心脏发育涉及一个线性心脏管形成, 心脏管循环, 和四个室间隔。这些复杂的过程在所有脊椎动物中都是高度保守的。小鼠胚胎心脏已被广泛用于心脏发育研究。然而, 由于其非常小的尺寸, 解剖老鼠胚胎心脏是技术上具有挑战性的。此外, 心脏室形成的可视化往往需要原位杂交, β-半乳糖苷酶染色使用 LacZ 报告小鼠, 或免疫染色的切片胚胎心脏。在这里, 我们描述了如何解剖小鼠胚胎心脏和直接可视化室形成的 Mlc-2v-td 番茄小鼠使用整个安装上皮荧光显微镜。利用这种方法, 可以直接检查心脏管的形成和循环, 四腔形成, 而无需进一步的实验操作小鼠胚胎。尽管本协议中使用了 Mlc-2v-td-tdato 记者敲门砖线, 但该协议可应用于其他心脏特定荧光记者转基因鼠标线。

在心脏发育过程中, MLC-2v 被认为是室腔规格17的最早标记。如图 1所示, 我们从 Mlc-2v-ttato 记者敲鼠标中分解了老鼠的整个胚胎和胚胎心脏, 并检查了 Mlc-2v-tdtoto 报告在心脏发育过程中的表达。在 Mlc-2v-td-tdato 记者敲不入小鼠中, 早在 e8.012 (图 2) 就通过显荧光整体贴装成像显示了发育中心脏中的本构 Tdtomto 表达 (<strong ...

Watch this Scientific Journal Video about Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence at JoVE.com

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

我们提出的方案, 以检查小鼠心脏的发展使用整个安装视网膜显微镜从心室特异性 Mlc-2v-t分解番茄记者敲入小鼠解剖小鼠胚胎。这种方法使我们能够直接想象小鼠心脏发育过程中心室形成的每个阶段, 而无需劳动密集型组织化学方法。

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

使用这种方法，我们可以直接检查心管的形成，循环，和全室形成，而无需进一步的实验操作小鼠胚胎。虽然，我们使用MLC-2v-tdTomato小鼠为例，这个简单的方法可以广泛用于其他荧光记者鼠标线。该协议的主要优点是非常简单，不需要任何复杂的技术或设备。它可以在常规实验室设置下执行。雌性小鼠与雄性小鼠交配后，MLC-2v-tdTomato 8至10周大，每天早上检查大坝有阴道塞。放置怀孕的水坝，安乐死在不同的日子后，在上部的位置，并喷洒腹部与70%乙醇。使用锋利的手术剪刀和钳子通过皮肤和腹壁的切口打开腹腔。在腹腔的后侧找到双边子宫角后，使用锋利的手术剪刀和钳子将整个子宫分开，小心地切开两侧的卵巢上方。将整个解剖子宫放在一个10厘米的培养皿中，并加有冰冷的PBS。使用锋利的手术剪刀和钳子，小心地沿着子宫角分离每个羊膜囊。使用钳子将每个胚胎移植到充满冰冷 PBS 的 35 毫米培养板中。使用锋利的手术剪刀和钳子，打开羊膜囊，通过切断脐带暴露每个胚胎，然后尽可能修剪出额外的胚胎组织，而不会损坏胚胎。在用光纤显微镜照明器解剖显微镜下，切断胚胎的头部，将其转移到1.5毫升的管中，并配以100微升的缓冲器 A，供以后进行基因分型，与荧光成像结果相关。使用精细钳子，打开胚胎的胸部。用锋利的手术剪刀和钳子，将心脏从肺和血管中去除。使用钳子将解剖的胚胎心脏转移到带PBS的35毫米培养板中。将带小鼠胚胎心的板放在荧光解剖显微镜下。使用精细钳子定位胚胎心脏，使发育中的心室面对检查员。在亮场模式下使用 0.63 倍目标调整焦点。拍摄亮场曝光并捕获多个图像。要可视化 tdTomato 表达式，请关闭光纤显微镜照明器并设置红色荧光滤镜。必要时重新调整图像对焦，调整亮度和对比度，拍摄红荧光曝光，并捕获多个图像。对于胚胎基因分型，在100摄氏度的缓冲器中将先前分离的头部样本煮沸30分钟。在 11，360 倍 G.将 20 微升中分的超量液转移到新的 1.5 毫升管中，并混合 20 微升，在 11，360 倍时离心两分钟。使用 4.5 微升的混合上流液作为 DNA 模板，并将其与 10 个微摩尔特异性前向和反向底塑剂的 0.5 微升、10 微升的 2x 预混合聚合酶和反应缓冲液，然后将水添加到 20 微升的总体积中。运行手稿中描述的 PCR。将完成的反应加载到 1%agarose 凝胶上的 100 基对 DNA 梯子上，并在 1x TAE 缓冲液中以 140 伏运行 25 分钟。将完成的凝胶放在紫外线透光器上，然后打开紫外光以识别DNA波段。MLC-2v被认为是心脏发育过程中心室规格最早的标记。在MLC-2v-tdTomato记者敲击小鼠中，E8.0的线性心管中tDTomato的表达相对较弱，在E8.5时变得更强，如使用胚胎全安装脱光成像所示。使用解剖心脏的全安装表皮成像，在小鼠心脏发育过程中可以轻松确定心室的形成。在E10.5的整个小鼠胚胎解剖的心脏中，MLC-2v-tdTomato记者的表情显示在心脏的心室部分，而不是流入道、流出区或未来的腹水。在E12.5到E13.5，tdTomato记者只在四室心脏的心室中表达。E16.5的解剖小鼠胚胎显示了类似的心室特异性表达模式。经过整个成像后，使用两组PCR基因分型基质确定胚胎的基因型，确认携带tdTomato敲击等位基因、异质和野生型胚胎的同源性胚胎。此方法非常简单且易于执行。关键的一步是解剖胚胎心脏。在心脏发育过程中了解心脏解剖学是成功将心脏从整个胚胎中分离出所必需的。

Research

JoVE Journal

Developmental Biology

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

优化<em&gt;前OVO</em&gt;鸡胚培养，以发展的高级阶段

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

科研

发育生物学

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic 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 ...

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

运用<em&gt;离体</em&gt;全胎儿各器官的直立滴文化的过程中鼠标研究器官发育过程

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

器官培养和鼠标沃尔弗导管的全山免疫染色

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

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

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 ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

培养小鼠卵巢的全贴装免疫荧光和卵泡定量研究

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

脊髓培养法研究胚胎鸡组织的发展

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

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

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 ...

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

达尼奥胚胎发生过程中的上眼硫可视化

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

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