Name: whole-mount in situ hybridization in zebrafish embryos and tube formation assay in ipsc-ecs to study the role of endoglin in vascular development
Uploaded: 2020-05-28T13:00:00
Description: Watch this Scientific Journal Video about Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development at JoVE.com

这项工作得到了国家干细胞转化研究重点研究发展计划（资助编号2016YFA0102300（致杨俊）的资助;中国自然科学基金[资助号81870051，81670054（给杨俊）];CAMS医学创新基金[授予编号2016-I2M-4-003（致杨俊）];PUMC青年发现和中央大学基础研究基金[授予编号2017310013（方周）]。

所有动物实验均获得浙江大学医学院研究伦理委员会批准。
1. 全装原位杂交
<ol><li>斑马鱼线畜牧业和繁殖<ol>
<li>在循环水产养殖系统中以26-28°C喂食和养殖所有成年斑马鱼，每天以14小时光/10小时暗循环。使用 AB（野生类型）斑马鱼线执行以下操作。</li>
		<li>在繁殖箱底部放一层鹅卵石作为卵子的庇护所。将父母鱼的比例为2：1（即，两只雌性鱼和一只雄性鱼）放入每个盒子...

<ol>
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W., 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=Endoglin+controls+blood+vessel+diameter+through+endothelial+cell+shape+changes+in+response+to+haemodynamic+cues.">Endoglin controls blood vessel diameter through endothelial cell shape changes in response to haemodynamic cues.</a> Nature Cell Biology. 19 (6), 653-665 (2017).</li><li>Zhang, 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=Endoglin+is+a+conserved+regulator+of+vasculogenesis+in+zebrafish+-+implications+for+hereditary+haemorrhagic+telangiectasia.">Endoglin is a conserved regulator of vasculogenesis in zebrafish - implications for hereditary haemorrhagic telangiectasia.</a> Bioscience Reports. 39 (5), (2019).</li><li>Lawera, 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=Role+of+soluble+endoglin+in+BMP9+signaling.">Role of soluble endoglin in BMP9 signaling.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (36), 17800-17808 (2019).</li><li>Gallardo-Vara, E., Tual-Chalot, S., Botella, L. 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该协议在HHT患者的iPSC-ECs上应用了改进的全位RNA分析平台，用于斑马鱼和管状检测。传统的ISH方法需要更长的实验周期和额外的实验步骤。该协议有一些重要的改进，分别使用独立双Z探头和iPSC从HHT患者身上获得，分别应用于WISH测定和管状。与传统 ISH 相比，这些改进对于提高检测灵敏度至关重要。独特的设计探头和目标信号放大允许检测很少表达的转录（aplnra是24个hpf胚胎中表达不良的基?...

在HHT患者中eng报告了eng（CD105）的单突变。突变导致EC增殖增加和减少流动介导EC伸长11，2。2也曾报道，ENG缺乏减少内皮标记表达（即，kdrl，cdh5，嘿2）在斑马鱼cdh53。Endoglin，主要以内皮细胞表示，是一种跨膜糖蛋白，是转化生长因子β（TGF-+）家庭成员的共同受体。它引导BMP9结合在细胞表面，以调节下游基因，包括Id1表达，诱导干细胞分化到ECs4。因此，eng基因在血管生成和人类血管疾病55、66中起着重要的作用。我们以前曾研究过苯丙烯酶在斑马鱼胚胎中撞击血管形成的影响，然后分析从具有eng突变7的HHT患者那里获得的iPSCs衍生的ECs。该协议演示了ENG缺乏对内皮祖体标记表达和管的形成的影响，这是一种测量体外血管生成的方法。
为了研究英语空间和时间表达，WISH用于检测体内基因表达8。原位杂交 （ISH） 是一种使用带有目标核酸（DNA 或 mRNA）补充序列的标签探头来检测和可视化固定试样中的目标核酸混合物的方法。该过程放大体内的基因表达信号，用于通过显微镜检测基因的表达。WISH已广泛应用于各种模型动物，特别是在斑马鱼9。它还用于获取以下数据：1）基因空间/时间表达模式，提供有关基因功能和分类的信息;和2）专门表示基因标记，用于高通量药物或突变筛选10。
色谱探针容易降解与传统的染色体原位杂交（CISH），导致高背景噪声和非特异性信号11，12。11,WISH方法使用两个独立的双Z探针，它们被设计成杂交以靶向RNA序列。每个探针包含一个与目标RNA互补的18-25序列和14个基尾序列（概念化为Z）。目标探头用于对（双 Z）。两个尾部序列共同形成前置放大器的 28 个基本混合站点，其中包含 20 个放大器的绑定位点。放大器，反过来，包含20个结合点的标签探头，理论上可以产生高达8，000个标签为每个目标RNA序列。
这一先进技术有助于同时进行信号放大和背景抑制，实现单分子可视化，同时保持组织形态13。WISH方法的进一步修改是基于先前的研究14，包括额外的固定和DAB染色。此处提供了改进的 WISH 协议，即使目标 RNA 部分减少或降解，也可以工作。优点包括可在 24 小时内完成，无需无 RNase 条件。信号也可以通过多个目标的多个通道同时检测，结果与不同高通量自动化平台的结果一致且兼容。
动物模型的结果没有必要反映人类发生的相同现象。ENG包含两对保守的半胱氨酸，C30-C207和C53-C182，在孤儿区形成不硫化桥梁。为了进一步研究eng在HHT患者中的作用，从HHT患者衍生的iPSC的管状检测已经在没有/有eng突变的细胞中进行（Cys30Arg，C30R）15。15在久保田等人首次报告管状实验16后，该测定已经以多种方式得到开发。它已被用于识别血管生成或抗血管生成因子，定义血管生成的信号通路，并确定调节血管生成的基因17。
在提供患者衍生的iPSC-ECs之前，研究人员主要使用培养的酶来研究血管原体16。然而，对于内皮细胞来说，用病毒转导外源基因是一项技术挑战，因为ECs可以经历的通道数量有限。这是因为几乎没有足够的细胞材料从人类血管收集，无论是从手术或匹配批准的捐赠者。随着iPSC生成技术的发明，由iPSC衍生的人类EC可以可靠地用于体外实验，如前18日报道。
使用病毒转导的ECs的数量和通道可能足以信号研究，但对于功能研究，最好产生突变多能干细胞系，（或iPSCs或CRISPER/Cas9目标hESCs），然后将它们区分成ECs，用于使用管形测定19的血管生成研究。管的形成可用于评估具有突变的内皮细胞的功能。该协议还描述了α-滑动血管生成板上的管状，这是一种简单、经济、可重复的方法。
下面的协议提供了一个可靠和系统的方法，用于研究特定基因在血管形成中的作用，以及体内表达模式和体外功能定量的细节，用于模拟人类疾病。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#181;-Slide Angiogenesis</td>
			<td>ibidi</td>
			<td>81506</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Amp 1-FL</td>
			<td>ACD</td>
			<td>SDS 320852</td>
			<td>Signal Amplification</td>
		</tr>
		<tr>
			<td>Amp 2-FL</td>
			<td>ACD</td>
			<td>SDS 320853</td>
			<td>Signal Amplification</td>
		</tr>
		<tr>
			<td>Amp 3-FL</td>
			<td>ACD</td>
			<td>SDS 320854</td>
			<td>Signal Amplification</td>
		</tr>
		<tr>
			<td>Amp 4 Alt B-FL</td>
			<td>ACD</td>
			<td>SDS 320856</td>
			<td>Signal Amplification</td>
		</tr>
		<tr>
			<td>Corning Matrigel Matrix</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Growth factor-reduced Matrigel</td>
		</tr>
		<tr>
			<td>DEPC</td>
			<td>Sigma</td>
			<td>D5758</td>
			<td>RNAase-free Water</td>
		</tr>
		<tr>
			<td>Human Endothelial-SFM</td>
			<td>Thermofisher</td>
			<td>11111044</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>30525-89-4</td>
			<td>Fixed embryos</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>30525-89-4</td>
			<td>Fixed Cells</td>
		</tr>
		<tr>
			<td>Protease K</td>
			<td>ACD</td>
			<td>SDS 322337</td>
			<td>Digest tissue</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Sigma</td>
			<td>6132-04-3</td>
			<td>SSCT solution: Wash Buffer</td>
		</tr>
		<tr>
			<td>VEGF-165</td>
			<td>STEMCELL Technologies</td>
			<td>78073</td>
			<td>Growth factor</td>
		</tr>
	</tbody>
</table>

whole-mount in situ hybridization in zebrafish embryos and tube formation assay in ipsc-ecs to study the role of endoglin in vascular development

血管发育由特定基因的连续表达决定，可在不同发育阶段对斑马鱼进行原位杂交测定进行研究。为了研究在遗传性出血性特朗吉性（HHT）发展过程中，在血管形成中的作用，利用斑马鱼中拟定的定向击倒来研究其时间表达和相关功能。在这里，全位原地RNA杂交（WISH）用于分析斑马鱼胚胎中的eng及其下游基因。此外，在HHT患者诱导多能干细胞分化内皮细胞（iPSC-ECs;具有eng突变）中进行管状检测。使用整量原位杂交的特定信号放大系统 – 与传统方法相比，WISH 可提供更高的分辨率和更低的背景效果。为了获得更好的信号，在探头杂交后，固定后时间将调整为30分钟。由于荧光染色在斑马鱼胚胎中不敏感，因此在这里被二氨基丁（DAB）染色所取代。在此协议中，含有eng突变的HHT患者衍生iPSC系分化成内皮细胞。在37°C下涂了30分钟的基底膜基质后，iPSC-EC作为单层播种成井，并保持在37°C3小时。然后，使用微观图像计算管长和分支数。因此，通过这种改进的WISH协议，表明减少的英性表达会影响斑马鱼胚胎的内皮祖祖形成。这进一步证实了使用从HHT患者的iPSC-ECs的管形测定。这些测定证实了eng在早期血管发育中的作用。

整个原位杂交基于类似于荧光共振能量传递的原理。它旨在提高斑马鱼ISH的灵敏度和特异性，并在不影响背景信号的情况下放大目标特异性信号。
在24个hpf斑马鱼胚胎中，在后侧基静脉（PCV）、分段血管（ISV）和血岛3中高度表达。在传统的色原原位杂交（CISH）中，很难控制染色时间，在一些地区出现?...

Watch this Scientific Journal Video about Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development at JoVE.com

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

本文介绍的是一种在斑马鱼和管状细胞中对斑马鱼进行全位RNA杂交分析的协议，在患者诱导的多能干细胞衍生内皮细胞中进行管状分析，以研究内索林在血管形成中的作用。

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

与传统方法相比，全安装、原位杂交、WISH提供了高分辨率和低背景结果。此外，U-滑动板改善焦距，使用较少的愿望可应用于小鼠胚胎，果蝇胚胎和其他组织难以处理。管形成测定可应用于干细胞分化和激素细胞。愿望使理解突变校正的功能意义变得容易。演示这个程序的人是我实验室的研究助理王勇。在显微镜下工作，使用FemtoJet系统将两个莫福利诺斯的南图和500个MMNA象形图注射到单细胞胚胎中。多次练习莫菲诺的微注射，直到你能成功地将其注射到细胞中，并保持卵子的完整性。孵育受精卵，直到它们被消减，达到与预期实验相对应的发展状态。如手稿表之一所述。接下来，使用塑料移液器将20至40个胚胎移植到1.5毫升管中。在室温下加入一毫升新鲜准备的4%PFA溶液。如稿中所述，在固定、清洗和脱水胚胎后，将胚胎放在底部用尼龙网的筛子中。通过按顺序清洗胚胎，在75%50%和25%甲醇各5分钟中水合物胚胎。用PBST清洗胚胎后，将50微升的蛋白酶K加入胚胎管中。消化后，取出蛋白酶K，用PBST清洗胚胎三次，每次五分钟。将50至100微升的混合靶探针加入每个管与胚胎。在40摄氏度下孵育胚胎过夜。如手稿所述，清洗胚胎后，使用4%的半甲醛在室温下固定胚胎30分钟。然后在室温下用2xSSCT溶液清洗胚胎3次，每次15分钟。卸下 SSCT 溶液，用 50 微升安培 1 将其更换。然后在40摄氏度下孵育胚胎。30分钟后，在室温下用2xSSCT溶液清洗胚胎3次，每次15分钟。卸下 SSCT 解决方案并添加 50 微升安培 2。孵育15分钟后，像之前一样清洗胚胎，加入50微升的安培3，轻轻敲开管子。在40摄氏度下孵育胚胎。30分钟后，如前面所述，清洗胚胎。然后添加 50 微升的 Amp 4 滴，并小心地敲开管子。然后在40摄氏度下孵育胚胎15分钟，然后用SSCT洗涤三次。准备胚胎进行成像后，使用 DAB 过氧化物酶基质套件染色标本。在一毫升蒸馏水中，每升50微升的彩色试剂：A、B和C。并很好地混合以获得完整的DAB工作液。将液体加入试样并盖上十分钟。彻底清洗标本后，使用光学显微镜对标本进行成像。要开始培养和控制HHT iPSCs，请将地下室膜基质添加到六个孔细胞培养板中，以覆盖井底。在37摄氏度下孵育板一小时。接下来，从井中去除用过的基底膜基质溶液，并在每口井中加入两毫升mTeSR 1介质。然后将从最后一个通道中收获的 iPSC 板到每井 6 个细胞的大约 1 倍。培养 iPSC 约四天后，用补充的 BEL 培养培养器交换细胞培养培养。生长细胞三天，产生中皮细胞。要扩大血管内皮细胞，用补充的BEL介质替换该介质四天。然后用相同的培养和培养的细胞再治疗三到四天。使用CD31 Dynabeads根据试剂盒指令净化成熟的血管内皮细胞。然后通过洗脱缓冲器收集CD31阳性细胞。在含有VEGF165、bFGF和FBS的EC-SFM介质中维护和扩展纯化的内皮细胞。要对内皮细胞进行板式，首先将每井10微升的基底膜基质添加到血管生成板中。在37摄氏度下孵育板30分钟。通过反复移液，收获内皮细胞，并在内皮生长介质2中重新分配。将50微升的细胞悬浮液加入每个井的凝固基质中，然后在37摄氏度下孵育细胞。经过三到五个小时的孵育，使用高分辨率显微镜评估内皮管的形成。捕获每个组至少 10 个区域的图像。检查整体管长、管号和分支点。进行CISH和希望，以确定在受精后24小时胚胎内内蛋白的表达。血源内皮标记和内皮祖细胞标记用于检查内皮沉默的效果。红色箭头指示这些标记（aplnrna 和 npr1a）的表达式显著减少的区域。内皮管形成测定在内皮蛋白突变体上进行，并控制 iPSC 衍生的内皮细胞。三小时后对管的形成进行评估和拍照。使用 ImageJ 软件对管长度、管号和分支进行量化。突变内皮细胞形成的分支比控制内皮细胞少。突变内皮细胞的分支在血管内皮生长因子刺激后显著增加。此方法也可用于从外周血生成 iPS。通过该方法阐明了内分泌在体外和体内血管形成中的作用。知道纠正突变可以改变血管生成，有可能移植回患者的潜在的干细胞治疗。

Research

JoVE Journal

Developmental Biology

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

多目标显色全安装<em&gt;原位</em&gt;在比较基因表达域杂交<em&gt;果蝇</em&gt;胚胎

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

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 ...

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 ...

Wholemount In Situ Hybridization for Astyanax Embryos

Wholemount In Situ Hybridization for Astyanax Embryos

阿斯蒂亚纳克斯胚胎的全原位杂交

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

斑马鱼原位杂交染色的基因分型与定量

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

斑马鱼胚胎和幼虫的整个山免疫性化学

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...