这项研究的灵感来自Levine等人的工作。描述了这一实验方法，并提供了非洲爪蟾血管发育的综合描述。我们感谢我们实验室的成员的投入。本研究得益于延世大学未来领先的2015年研究计划（2015年至22年）和国家科研基金会（NRF）的生物和医学技术开发计划（NRF），由科学，ICT与未来计划部NRF-2013M3A9D5072551）

所有实验均符合延世大学医学院动物保健和使用委员会批准的方案。 1. 非洲爪蟾热带胚胎的准备注意: 如前所述 ，制作了非洲爪蟾热带胚胎，稍作修改。 非洲爪蟾热带胚胎根据Nieuwkoop和Faber 11的表进行分期。 <ol><li> 诱导排卵 <ol><li>要预先注射，在交配之前一天，将一种青蛙（一只雄性和一只雌性）的人绒毛膜促性腺激素（hCG）注射到背部淋巴囊中。每只雌蛙每只灌注20只，每只雄性10只。在同一个水箱内一个男人和一个女人在一夜之间（超过18小时）。 </li><li>首先，第二天将200单位的hCG注射到预先灌注的女性和100单位的hCG中，以预先灌注男;被引导的女性将在约3小时内开始产卵，并且将在全天继续这样做。将雄性和雌性对保持在一起用于自然交配，或在单独的罐中进行体外受精 （步骤1.2）。 </li></ol></li><li> 受精 注意:鸡蛋可以通过自然交配或体外受精来受精。在自然交配中，鸡蛋被铺设时受精，因此受精时间不能控制。或者，可以将引发的雌性青蛙饲养在单独的罐中，并且可以收集未受精卵以用于体外受精 ，其同时产生数百个受精的胚胎。当在血管标记之前进行卵裂球靶向显微注射时，后一种方法是有用的。然而，在这种方法中，牺牲了男性。 <ol><li>自然交配<ol><li>在同一个坦克里留下原始的女性和男性对。男性将扣住f导致立即受精的鸡蛋。用玻璃或塑料移液器收集鸡蛋，并将其转移到培养皿中。为了防止鸡蛋粘在移液管上，用0.5％牛血清白蛋白（BSA）涂抹移液管的内表面。 </li></ol></li><li> 体外受精 （IVF） <ol><li>起动后，将起始的女性与男性分开。女性将在大约3小时内自发产卵。使用BSA涂层移液器将一些铺设的卵转移到培养皿中，并在显微镜下检查其质量。如果放置健康的蛋（透明颜料，弹性球形），准备从被涂的男性中剖析睾丸。 </li><li>使0.4％的甲磺酸三甲基氯化钠（MS222）进行安乐死，并将300μL注射到已注射的男性的背部淋巴囊中。大约10分钟后，男性将失去平衡感，并保持活力;确认通过用一个后腿夹住男性来安乐死一对钝镊子，观察无反应。 </li><li>解剖出两个睾丸，通过在无绒纸上快速敲打将其清洗干净，并将其转移到含有1 mL 60％Leibovitz L-15培养基（60％）的微管中，补充有10％胎牛血清（FBS）;详细的程序在其他地方描述10 。 </li><li>用杵轻轻剁碎睾丸以提取精子。在该浓度下（ 即，在1mL 10％FBS在60％L-15 中的 2次睾丸）中，几滴睾丸溶液（0.1mL）可以施用约三百个卵。剩余的精子可以在4°C密封管中储存，第二天用于IVF。 </li><li>将鸡蛋从女性挤压成小培养皿。将女性的上半部分倒在手掌上，将食指放在其后腿之间。用食指和另一只手伸展后腿，露出泄殖腔。触摸泄殖腔到培养皿。外套他用0.5％BSA的培养皿在IVF期间以单层均匀分布卵。 </li><li>向蛋中加入几滴（0.1 mL）含精液的L-15培养基。对于同步受精，轻轻地将精子溶液搅拌过菜。在室温下4分钟后，用0.01x改良巴斯氏盐水（MBS）填充培养皿，孵育10分钟，并用0.1x MBS替代溶液。受精卵将在室温约1小时内进行第一次裂解。 1×MBS是88mM NaCl，1mM KCl，2.5mM NaHCO 3，5mM HEPES，1mM MgSO 4和0.7mM CaCl 2 ，pH7.5。 </li></ol></li></ol></li><li> （可选）注射反义吗啉代寡核苷酸（MO）和/或mRNA <ol><li>为了研究感兴趣的基因对血管形成的影响，进行反义MO和/或mRNA的显微注射。 </li><li>对于这种实验，将1％MBS中2％L-半胱氨酸的鸡蛋冻干（pH 7.5-7.8）。在含有1％MBS中2％L-半胱氨酸的受精胚胎的培养皿中取代0.1x MBS。轻轻旋转盘子混合溶液。 注意:胚胎脱蛋白通常需要约5分钟。在此步骤中，经常监视鸡蛋，以确保其不会过度暴露于溶液中。如果鸡蛋过度暴露，就会失去弹性并变扁，这将导致异常发育和致死。 </li><li>用0.1x MBS洗涤去皮蛋5次。在0.1％MBS中溶解4％Ficoll进行显微注射。通常，在两细胞阶段，每个卵裂球注射4ng的MO和600pg的mRNA。注入一个细胞，仅在胚胎的一侧操纵基因表达。使用同一胚胎的未注射侧作为对照。 </li><li>作为基因操作特异性的另一个对照，注入相同量的对照MO（CoMO）或对照mRNA（ 例如， β-半乳糖苷酶mRNA）。 CoMO必须有相同的m作为基因特异性MO的分子量，不能靶向非洲爪蟾基因组中的任何基因。为了容易观察注射侧，使用荧光素标记的MO或共同注射示踪剂（ 如 EGFP mRNA）。 </li><li>将注射的胚胎留在4％Ficoll中2小时，然后将其转移到填充有0.1x MBS的新的60mm培养皿中。 </li></ol></li><li> 提高 <ol><li>在23℃升高胚胎。虽然通过使用温度低于或高于23°C（范围:16-26°C）可以减缓或加速发展速度，但建议在23°C下升温。 </li></ol></li><li> （可选）用药理试剂处理 <ol><li>对于药物操作，通过浴应用向发育中的胚胎施用药物。 </li></ol></li></ol> 2.制备DiI-AcLDL注射液<ol><li>使用标准微型仪制成锥形玻璃移液器操作面板拉拔器将硼硅酸盐玻璃管放在微量吸管拔出器中，并使用以下设置:压力，200;热，30;拉，30;速度120时间，200。 </li><li>将DiI-AcLDL储备溶液储存在4°C。取上清液注射溶液，但不要在管底沉淀的颗粒。玻璃移液管中的任何碎屑会干扰溶液的正确喷射。 </li><li>使用微型加载器，用适量的DiI-AcLDL溶液（〜5μL）填充准备好的玻璃移液管。 </li><li>用精细的镊子（55号）一点一滴地夹住玻璃移液器的尖端。设置压力控制的注射系统，以便每次喷射约5 nL的溶液; 50-60 nL的DiI-AcLDL溶液足以染色一个胚胎的血管。 </li><li>不要将DiI-AcLDL装入的吸管长时间放置在空气中，以避免干燥。保持移液管的尖端浸没在0.04％MS222在0.1X MBS溶液中。在将其注入胚胎之前（步骤4.2.1），检查是否喷出相同量的染料。 </li></ol> 3.注射设置<ol><li> Sylgard模具 <ol><li>为了制备小型Sylgard模具，将Sylgard碱和固化剂与重量比为10:1混合，用该混合溶液填充约一半的小培养皿（35mm或60mm皿），然后将其固化约室温48小时。 </li></ol></li><li> 麻醉 <ol><li>使用1x MBS（pH 7.5-8.0）中的0.04％MS222进行麻醉。当需要长时间麻醉（ 例如，在实时成像期间），使用0.1％MBS中的0.04％MS222来减少渗透不平衡。要调节pH值，加入几滴NaOH（〜20μL），用pH值纸检查pH值。 </li><li>等待转移的胚胎停止移动。触摸胚胎的背鳍，检查它们是否响应确认完成nesthesia。 </li></ol></li><li> 定位胚胎注射 <ol><li>用薄而锋利的刀片缩小硬化Sylgard模具的表面。做一个V形的凹陷，放置胚胎（ 见图2D ）。将麻醉的胚胎腹侧面向上倾斜（约30°）（ 图2D ）。 </li><li>用MS222溶液填充模具，并将胚胎置于模具中。 </li></ol></li></ol> DiI-AcLDL注射液<ol><li> 皮肤覆盖心脏的切口 <ol><li>准备两个针（Minutiens，0.10 mm）以切割心脏覆盖的皮肤。将每个针紧紧地固定在一个针架上;心脏应易于识别，因为它会脉动（ 图2A-2C ，粉红色）。 </li><li>用一个针刺穿胚胎的皮肤。将针插入心脏和皮肤之间的空间中的孔（ 图2C </strong&gt;，实线箭头）。不要穿刺心脏。将该引脚的插入部分置于切口的区域。 </li><li>通过轻轻地将固定在皮肤外部的另一个针轻轻地摩擦插入的针，进行线性切口。用两个针轻轻加宽这个切口，暴露心脏（ 图2B&#39;和2D&#39; ，箭头）。装有DiI-AcLDL的移液管现在可以直接接近心脏（ 图2D&#39; ）。 </li></ol></li><li> 注射 <ol><li>将装载的玻璃移液管的尖端插入心脏，并以约10次喷射脉冲注射50-60 nL的DiI-AcLDL溶液。不要注射过量的溶液，因为它会导致心脏破裂。检查注射后心脏是否继续拍打。每次注射后，检查是否喷出相同量的DiI-AcLDL;否则，移液管的尖端可能被堵塞（参见步骤2.5）。 </li></ol></li><li><stro恢复和视觉筛选<ol><li>将注射的胚胎转移到装有0.1x MBS的新培养皿中以进行恢复。 5-10分钟后，ad oles应恢复意识，开始行动。此时，标记的血管可被成像。建议在荧光显微镜下直观筛选成功标记的胚胎（ 图3A和3B ）。如果将不足量的DiI-AcLDL注射到胚胎中，则可以再次注射（ 图3C ）。 </li><li>丢弃标有其他器官的ad oles（ 图3D ）。继续，直到获得所需数量的成功标记的胚胎。 </li></ol></li><li> （可选）胚胎的固定 <ol><li>为了更全面的血管成像，使用共焦显微镜;固定标记的胚胎可能更容易共聚焦显微镜。首先用1×MB的0.04％MS222麻醉标记的胚胎S（pH 7.5-8.0），然后在1×磷酸盐缓冲盐水（PBS）中在4％多聚甲醛中固定1小时。固定的胚胎可以在4℃下储存几天;保护注射的样品免受光照，以避免DiI的漂白。 </li></ol></li></ol> DiI-AcLDL成像<ol><li> 立体成像 <ol><li>在荧光立体显微镜下拍照（ 图3 ）以评估脉管系统的整体形态。 </li><li>在1x PBS中融合1％琼脂糖固定胚胎或1×MBS用于活体成像。将琼脂糖倒入培养皿中，等到固化。在琼脂糖凝胶表面上形成一个浅凹痕，使其适合在躺在其侧面时成像的胚胎。用缓冲液（MS222麻醉的胚胎溶液或1x PBS固定胚胎）填充盘子。 </li></ol></li><li> 荧光显微镜（Rhodamine filter seT） <ol><li>由于DiI是绿色激发和红色发射的染料，使用用于罗丹明或光谱相关荧光团的标准滤光片的图像（在554nm处的最大激发和在571nm的发射）。将注射的胚胎置于琼脂糖模具上的凹槽中并拍摄照片（ 图3 ）。 </li></ol></li><li> 共焦显微镜 注意:为了更紧密地分析血管结构，使用共焦显微镜。 <ol><li>如果使用倒置显微镜，将胚胎置于玻璃底盘上。加入几滴1x PBS，并通过在顶部放置盖子将其固定。清除多余的PBS。如果活胚成像，使用麻醉的胚胎，并使用0.015％的MS222在0.1x MBS而不是PBS。 </li><li>对于低倍率成像，使用4X到10X的物镜来找到感兴趣的区域;后基底静脉（PCV）的分泌部位是调查发育性血管生成的有用区域（图4，虚线）框）。 <ol><li>使用红色发射荧光团（如罗丹明）的采集设置。 </li><li>通过成像胚胎的外侧半部来制作z堆叠的图像。首先，将焦点放在靠近物镜的侧面的PCV上，然后将焦点的下限设置在最接近物镜的血管聚焦的位置处。将被摄体的PCV的中间部分的位置设定为上限。形成整个胚胎的外侧一半。使用在采集设置（如x，y和z标度）上存储元数据的软件，因为它们需要计算容器长度。 </li><li>如果需要，使用瓦片扫描模式对胚胎的整个脉管系统进行成像。经验地确定水平和垂直瓦片的数量。 </li></ol></li><li>对于高放大倍率成像，按照上述步骤对PCV的长颈部分和4个最大的吻合脉管（ISV）进行成像（ 图4 ，虚线框）。适当调整堆栈数（z堆叠）和图块（平铺扫描）。 </li></ol></li></ol> DiI标记船只的定量注意:可以手动或使用软件追踪DiI标记的血管。我们使用"简单神经轴追踪器"，一个免费的ImageJ（NIH）插件，允许半自动跟踪管状结构，如血管和神经突12 。使用这个免费软件，可以计算以下参数。使用此软件的详细步骤在别处描述（https://imagej.net/Simple_Neurite_Tracer）12。以下，我们使用Tie2信号传导被抑制或增强的胚胎脉管系统的实例（ 图4A-4C ）。反义Tie2MO注射的胚胎表现出减少血管生成并缩短ISV（ 图4B ），而共注射组成型活性Tie2突变体mRNA（caTie2 mRNA）过度挽救了这种表型，导致了旺盛的ISV分支（ 图4C ）。 <ol><li> ISV长度 <ol><li>使用简单的神经轴示踪剂，计算每个追踪血管的长度。计算4个最常见的ISV的长度。 </li></ol></li><li> ISV分支机构 注意:在野生型胚胎中，只有少数短枝在第42阶段从最常见的ISVs发芽。 <ol><li>在简单的神经突征示踪剂中，追踪这些小血管，使ISV从其起源于主要分支。计算源自ISV的这些分支的以下参数。 </li><li>总支数<ol><li>一旦所有ISV和相关联的分支被追踪，计算总分支数（ 图5B-5D ）。 </li></ol></li><li>分行订单<ol><li>由于简单的神经轴跟踪记录每个分支的顺序（ 图5A ），计算e每个订单的分支数量。野生型胚胎中的大多数血管应为主要分支（ 即 ISV）。 </li></ol></li><li>静脉复杂指数（VCI） <ol><li>由于VCI是容器复杂性的有用参数，其给予较高阶分支更多的权重，使用图5A中的公式计算每个胚胎的VCI。例如，与对照相比，Tie2MO注射的胚胎具有较低的VCI（ 图5B和5C ），并且caTie2mRNA注射的胚胎具有较高的VCI（ 图5D ）。 </li></ol></li></ol></li></ol>

<ol>
	<li>Herbert, S. P., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+control+of+endothelial+cell+behaviour+during+blood+vessel+morphogenesis.">Molecular control of endothelial cell behaviour during blood vessel morphogenesis.</a> Nat Rev Mol Cell Biol. 12 (9), 551-564 (2011).</li><li>Augustin, H. G., Koh, G. Y., Thurston, G., Alitalo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+vascular+morphogenesis+and+homeostasis+through+the+angiopoietin-Tie+system.">Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system.</a> Nat Rev Mol Cell Biol. 10 (3), 165-177 (2009).</li><li>Lawson, N. D., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arteries+and+veins:+making+a+difference+with+zebrafish.">Arteries and veins: making a difference with zebrafish.</a> Nat Rev Genet. 3 (9), 674-682 (2002).</li><li>Levine, A. J., Munoz-Sanjuan, I., Bell, E., North, A. J., Brivanlou, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+labeling+of+endothelial+cells+allows+in+vivo,+continuous+characterization+of+the+vascular+development+of+Xenopus+laevis.">Fluorescent labeling of endothelial cells allows in vivo, continuous characterization of the vascular development of Xenopus laevis.</a> Dev Biol. 254 (1), 50-67 (2003).</li><li>Yang, 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=Calmodulin+Mediates+Ca2+-Dependent+Inhibition+of+Tie2+Signaling+and+Acts+as+a+Developmental+Brake+During+Embryonic+Angiogenesis.">Calmodulin Mediates Ca2+-Dependent Inhibition of Tie2 Signaling and Acts as a Developmental Brake During Embryonic Angiogenesis.</a> Arterioscler Thromb Vasc Biol. 36 (7), 1406-1416 (2016).</li><li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+32-cell-stage+Xenopus+embryo.">Fates of the blastomeres of the 32-cell-stage Xenopus embryo.</a> Dev Biol. 122 (2), 300-319 (1987).</li><li>Elliott, K. L., Houston, D. W., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+Xenopus+laevis+tissues+to+determine+the+ability+of+motor+neurons+to+acquire+a+novel+target.">Transplantation of Xenopus laevis tissues to determine the ability of motor neurons to acquire a novel target.</a> PLoS One. 8 (2), 55541 (2013).</li><li>Grainger, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+tropicalis+as+a+model+organism+for+genetics+and+genomics:+past,+present,+and+future.">Xenopus tropicalis as a model organism for genetics and genomics: past, present, and future.</a> Methods Mol Biol. 917, 3-15 (2012).</li><li>Weisgraber, K. H., Innerarity, T. L., Mahley, R. W. <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+lysine+residues+of+plasma+lipoproteins+in+high+affinity+binding+to+cell+surface+receptors+on+human+fibroblasts.">Role of lysine residues of plasma lipoproteins in high affinity binding to cell surface receptors on human fibroblasts.</a> J Biol Chem. 253 (24), 9053-9062 (1978).</li><li>Showell, C., Conlon, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egg+collection+and+in+vitro+fertilization+of+the+western+clawed+frog+Xenopus+tropicalis.">Egg collection and in vitro fertilization of the western clawed frog Xenopus tropicalis.</a> Cold Spring Harb Protoc. 2009 (9), 5293 (2009).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus laevis (Daudin). , (1956).</li><li>Longair, M. H., Baker, D. A., Armstrong, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+Neurite+Tracer:+open+source+software+for+reconstruction,+visualization+and+analysis+of+neuronal+processes.">Simple Neurite Tracer: open source software for reconstruction, visualization and analysis of neuronal processes.</a> Bioinformatics. 27 (17), 2453-2454 (2011).</li><li>Marshak, S., Nikolakopoulou, A. M., Dirks, R., Martens, G. J., Cohen-Cory, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-autonomous+TrkB+signaling+in+presynaptic+retinal+ganglion+cells+mediates+axon+arbor+growth+and+synapse+maturation+during+the+establishment+of+retinotectal+synaptic+connectivity.">Cell-autonomous TrkB signaling in presynaptic retinal ganglion cells mediates axon arbor growth and synapse maturation during the establishment of retinotectal synaptic connectivity.</a> J Neurosci. 27 (10), 2444-2456 (2007).</li><li>Cha, H. 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=Evolutionarily+repurposed+networks+reveal+the+well-known+antifungal+drug+thiabendazole+to+be+a+novel+vascular+disrupting+agent.">Evolutionarily repurposed networks reveal the well-known antifungal drug thiabendazole to be a novel vascular disrupting agent.</a> PLoS Biol. 10 (8), 1001379 (2012).</li><li>Ny, 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=A+transgenic+Xenopus+laevis+reporter+model+to+study+lymphangiogenesis.">A transgenic Xenopus laevis reporter model to study lymphangiogenesis.</a> Biol Open. 2 (9), 882-890 (2013).</li><li>Bussmann, 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=Arteries+provide+essential+guidance+cues+for+lymphatic+endothelial+cells+in+the+zebrafish+trunk.">Arteries provide essential guidance cues for lymphatic endothelial cells in the zebrafish trunk.</a> Development. 137 (16), 2653-2657 (2010).</li><li>Li, X. M., Hu, Z., Jorgenson, M. L., Slayton, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+levels+of+acetylated+low-density+lipoprotein+uptake+and+low+tyrosine+kinase+with+immunoglobulin+and+epidermal+growth+factor+homology+domains-2+(Tie2)+promoter+activity+distinguish+sinusoids+from+other+vessel+types+in+murine+bone+marrow.">High levels of acetylated low-density lipoprotein uptake and low tyrosine kinase with immunoglobulin and epidermal growth factor homology domains-2 (Tie2) promoter activity distinguish sinusoids from other vessel types in murine bone marrow.</a> Circulation. 120 (19), 1910-1918 (2009).</li></ol>

作者没有什么可以披露的。

这里提出的方案首先由Ali H.Brivanlou及其同事开发，以调查非洲爪蟾 4的血管形成过程中的发育事件，但如本手稿所示，可用于其他小动物。将染料注入心脏很容易进行，整个血管网络可以在荧光解剖显微镜下以及共聚焦显微镜下成像。如果染料在血管发育过程中注入心脏，血管生长和分支的动力学可以实时成像4 。使用明确定义的静脉网络，特别是PCV和最流行的ISV，可以定量评估遗传或环境扰动对血管生成的影响。 该协议中最关键的一步是注入DiI-AcLDL（步骤4）。我们提供成功和不成功注射的实例（ Fi3）。成功注射的胚胎应在荧光显微镜下进行筛选，如步骤4.3所述。如果PCV在注射后30分钟不可见，则不能注射足够的DiI-AcLDL（ 图3C ）。不成功注射的胚胎可能被麻醉并再次注射。在某些情况下，DiI-AcLDL可能被注入不正确的位置，在这种情况下，其他器官将被标记（ 图3D ）。丢弃注射不正确的胚胎。 成功注射胚胎的血管在注射后不久就可见，荧光持续数小时。因此，如果在形成ISV之前进行注射，则其发育可以在活胚中成像，为实时和在体内研究心血管系统的发展提供了有力的工具。因为非洲爪蟾已被成功地用作屏幕血管的模型在哺乳动物14中的破坏剂，这里描述的实验方法可能与药理学扰动实验相结合，并用作筛选平台来寻找增强或抑制血管发生的药物。 血管可以通过遗传工具以及这里描述的基于染料的标记方法来显现。例如，在细胞类型特异性启动子控制下表达荧光蛋白的转基因非洲爪蟾 15或斑马鱼16能够进行血液和淋巴管的活体成像，甚至可以区分动脉16和静脉16 。尽管遗传方法是优越的并且产生更一致的标记效率，但是大多数实验室容易获得基于染料的方法，而无需获得这种基因工程的动物。在非洲爪蟾中 ，DiI-AcLDL的经血灌注导致了标记大多数内皮细胞4 ，动脉和静脉在荧光图像中是不可区分的。有趣的是，番茄凝集素荧光素选择性地标记小鼠的动脉，当共同注射DiI-AcLDL时，可以区分动脉和静脉。虽然该应用未在其他组织或动物中进行测试，但这为调查非洲爪蟾动脉和静脉的动态发展提供了有希望的方向。

血管生成，从新出生的内皮细胞形成新血管，血管发生，从先前存在的血管形成新血管，是形成胚胎脉管系统的两个不同的过程。这些过程中的任何调节都会导致各种心脏疾病和血管的结构异常。此外，肿瘤生长与不受控制的血管生长有关。因此，血管发生和血管生成的分子机制是密切研究的对象2 。 非洲爪蟾和斑马鱼是有吸引力的脊椎动物模型，用于血管发生和血管生成研究，原因有几个。首先，他们的胚胎很小;因此，对整个脉管系统进行成像是比较容易的。其次，胚胎发育很快;整个脉管系统只需要几天的时间才能发展，在此期间，发展中的血管<html>可以成像。第三，容器形成之前和期间的遗传和药物干预是容易进行的，例如通过将反义吗啉代核苷酸（MO）显微注射到发育中的胚胎中或通过药物3,4,5的浴中应用。 非洲爪蟾对斑马鱼的独特优势是可以进行胚胎操作，因为非洲爪蟾遵循定型的全息切割，胚胎命运图很好地定义6 。例如，可以通过在两细胞阶段向一个细胞注射反义MO来产生仅遗传操纵一个侧面的胚胎。还可以将心原子从一个胚胎移植到另一个胚胎，以确定该基因是否通过细胞内在或外在机制发挥其功能ass ="xref"&gt; 7。虽然这些技术主要是在非洲爪蟾属中开发的，它是异源四倍体，因此不适用于遗传研究，但它们可以直接应用于热带非洲爪蟾 ，这是一种密切相关的二倍体物种。 在活非洲爪蟾胚胎中可视化脉管系统的一种方法是注射荧光染料以标记血管。用荧光分子如DiI标记的乙酰化低密度脂蛋白（AcLDL）是非常有用的探针。与未乙酰化LDL不同，AcLDL不与LDL受体9结合，但被巨噬细胞和内皮细胞吞噬。将DiI-AcLDL注射到活体动物的心脏中导致内皮细胞的特异性荧光标记，并且可以通过荧光显微镜在活的或固定的胚胎中成像整个脉管系统。 在这里，我们在非洲爪蟾中使用DiI-AcLDL进行血管显像和定量的详细方案（ 图1 ）。我们提供了关键的实践要点，例如成功和不成功的实验。此外，我们提供了一种用于定量分析血管复杂性的直接方法，可用于评估遗传和环境因素对血管网络形成的影响。

<table border="0" cellpadding="0" cellspacing="0" width="539">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">35mm Petri dish</td>
			<td style="width:113px;">SPL</td>
			<td style="width:93px;">10035</td>
			<td style="width:180px;">Sylgard mold frame</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">60mm Petri dish</td>
			<td style="width:113px;">SPL</td>
			<td style="width:93px;">10060</td>
			<td>Embryo raising tray</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Borosilicate Glass</td>
			<td style="width:113px;">Sutter instrument</td>
			<td style="width:93px;">B100-50-10</td>
			<td>Needle for injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">BSA</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">A3059-10G</td>
			<td>Coating reagent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">CaCl2</td>
			<td style="width:113px;">D.S.P.GR Reagent</td>
			<td style="width:93px;"></td>
			<td>0.1X MBS component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Coverslip</td>
			<td style="width:113px;">Superior</td>
			<td style="width:93px;">HSU-0111520</td>
			<td>For confocal imaging</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">DiI-AcLDL</td>
			<td style="width:113px;">Thermo Fisher Scientific</td>
			<td style="width:93px;">L3484</td>
			<td>Vessel staining solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">FBS</td>
			<td style="width:113px;">Hyclone</td>
			<td style="width:93px;">SH.30919.02</td>
			<td>For storage of testis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Fiber Optical Illuminator</td>
			<td style="width:113px;">World Precision Instruments</td>
			<td style="width:93px;">Z-LITE-Z</td>
			<td>Light</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Ficoll</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">F4375</td>
			<td>Injection buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Flaming/Brown Micropipette Puller</td>
			<td style="width:113px;">Sutter instrument</td>
			<td style="width:93px;">P-97</td>
			<td>Injection needle puller</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Forcep</td>
			<td style="width:113px;">Fine Science Tool</td>
			<td style="width:93px;">11255-20</td>
			<td style="width:180px;">For embryo hatching and 
			needle tip cutting</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Glass Bottom dish</td>
			<td style="width:113px;">SPL</td>
			<td style="width:93px;">100350</td>
			<td>For confocal imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">hCG</td>
			<td style="width:113px;">MNS Korea</td>
			<td style="width:93px;"></td>
			<td>For priming of frogs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">HEPES</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">H3375</td>
			<td>Buffering agent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Incubator</td>
			<td style="width:113px;">Lab. Companion</td>
			<td style="width:93px;">ILP-02</td>
			<td>For raising embryos</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">KCl</td>
			<td style="width:113px;">DAEJUNG</td>
			<td style="width:93px;">6566-4400</td>
			<td>MBS component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">L15 medium</td>
			<td style="width:113px;">Gibco</td>
			<td style="width:93px;">11415-114</td>
			<td>For storage of testis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">L-cysteine</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">168149-100G</td>
			<td>De-jellying reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">MgSO4</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">M7506</td>
			<td>MBS component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Microtube</td>
			<td style="width:113px;">Axygen</td>
			<td style="width:93px;">MCT-175-C-S</td>
			<td>For storage of testis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">MS222</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">E10521</td>
			<td>Anesthetic powder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">NaCl</td>
			<td style="width:113px;">DAEJUNG</td>
			<td style="width:93px;">7647-14-5</td>
			<td>MBS component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">NaOH</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">S-0899</td>
			<td>pH adjusting reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Paraformaldehyde</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">P6148</td>
			<td>Fixatives</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">PBS</td>
			<td style="width:113px;">BIOSESANG</td>
			<td style="width:93px;">P2007</td>
			<td>Buffer for imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">pH paper</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">P4536-100EA</td>
			<td>For confirming pH</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">PICO-LITER INJECTOR</td>
			<td style="width:113px;">Waner instruments</td>
			<td style="width:93px;">PLI-100A</td>
			<td>For injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Pin</td>
			<td style="width:113px;">Pinservice</td>
			<td style="width:93px;">26002-10</td>
			<td>For incision</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Pinholder</td>
			<td style="width:113px;">Scitech Korea</td>
			<td style="width:93px;">26016-12</td>
			<td>For incision</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Precision Stereo Zoom Binocular Microscope</td>
			<td style="width:113px;">World Precision Instruments</td>
			<td style="width:93px;">PZMIII</td>
			<td>For visual screening</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:153px;">Standard Manual Control Micromanipulator&#160;</td>
			<td style="width:113px;">Waner instruments</td>
			<td style="width:93px;">W4 64-0056</td>
			<td>For microinjection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">SYLGARD 184 Kit</td>
			<td style="width:113px;">Dow Corning</td>
			<td style="width:93px;"></td>
			<td>For DiI injection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Transfer pipette</td>
			<td style="width:113px;">Korea Ace Scientific Co.</td>
			<td style="width:93px;">YM.B78-400</td>
			<td>For eggs and 
			embryo collection</td>
		</tr>
	</tbody>
</table>

visualization and quantitative analysis of embryonic angiogenesis in xenopus tropicalis

血管在全身供给氧气和营养物质，血管网络的形成受到严格的发展控制。血管的有效体内可视化及其复杂性的可靠定量是了解血管网络的生物学和疾病的关键。在这里，我们提供了一个详细的方法，用市售的荧光染料，人血浆乙酰化低密度脂蛋白DiI复合物（DiI-AcLDL）可视化血管，并量化他们在热带非洲爪蟾的复杂性 。可以通过将DiI-AcLDL简单注入胚胎的跳动心脏来标记血管，并且在整个胚胎中的血管可以在活的或固定的胚胎中成像。通过靶向显微注射核酸和/或药理试剂的浴液应用基因扰动，基因或信号传导途径对血管发育的作用可以揭示在一个星期内不参与复杂的遗传工程动物。由于非洲爪蟾定义良好的静脉系统及其刻板血管生成，预先存在的血管发芽，血管复杂性可以在扰动实验后有效量化。这个相对简单的协议应该作为心血管研究领域的一个易于访问的工具。

实验时间表（图1和图2） 受精后不久，可以进行靶向显微注射以调节基因表达。例如，可以注射特异性结合内源性Tie2mRNA的起始密码子的反义MO，通过空间位阻抑制Tie2靶mRNA的翻译。 MO可以与荧光素缀合，以便成功注射胚胎的容易的目视筛选。或者，示踪剂mRNA（ 例如，编码EGFP的mRNA）可以与MO共注射。另外，药物可以通过孵化后的浴液治疗（早期尾巴阶段，NF阶段26周围）。将药物溶解于0.1x MBS，并将其加入胚胎。对于早期治疗，胚胎可以用一对镊子从膜中释放出来。 DiI-AcLDL可以注射到33/34级周围的心脏中，以调查血管形态的动态但通常我们在37/38或更高阶段注射（ 图2 ，彩色区域）。 成功和不成功注射的典型例子（步骤4.3）（图3） 立体成像适用于视觉筛查。如果将足够量的DiI-AcLDL注射到心脏中，PCV应在荧光立体镜下立即可见（ 图3A和3B ）。注射胚胎不足或不正确易于识别（ 图3C和3D ）。具有不足量的DiI-AcLDL的胚胎（ 图3C ）可以用相同的染料再次注射，直到容器清晰可见。图像成功地在共焦显微镜下注射胚胎，活或固定后。 后期发展或基底静脉（PCV）和交配静脉（ISV） PCV从rostal到尾部的方向延伸，这个延伸在第37/38号阶段结束。在前后波中，单核细胞病毒从PCV背部出现。这种ISV形成浪潮从36阶段开始，到40/41阶段结束; ISV继续生长并变得轻微分支直到阶段43（ 图3A和3B ）。 Tie2信号控制发育ISV分支（图4） PCV和ISV以刻板印象发展，其发展受到严格控制（ 图4A ）。从PCV形成的ISV可以描述为血管发生，因此它是研究血管生成的分子机制的有用模型。介绍我们最近研究的结果该发育信号抑制ISV中的Tie2信号以限制它们的分支5 。通过反义MO敲低Tie2信号传导减少ISV的长度和复杂性（图4B），并表达组成型活性形式的Tie2（caTie2）引起过度的ISV分支（ 图4C ）。 ISV复杂度的量化（图5） 除了ISV的长度和分支数之外，可以计算"静脉复杂度指数"（VCI）以评估其复杂性（ 图5A ）。我们采用了Cohen-Cory及其同事开发的"复杂性指标"，旨在量化神经元轴突或树突状乔木的复杂性。在这个指标中，具有更高阶分支的胚胎比具有相同数量的总麸皮的胚胎得到更高的分数但更多的是低阶分支。因此，较高的VCI表明该胚胎具有更复杂的静脉网络。 "简单神经轴追踪器"是有用的软件来跟踪各个分支的顺序和长度。还可以生成"渲染路径"，这是可视化脉管系统的整体架构（ 图5B-5D ，右图）的有用方式。 <img alt="图1" src="/files/ftp_upload/55652/55652fig1.jpg" /> 图1:实验的时间线。在受精当天，可以在NF阶段1（st1）或2（st2）下将受体卵中的吗啉代（MO），DNA，RNA或蛋白质注射入受精卵中。如果只注射到st2的一个卵裂球，注射的试剂只会影响一个侧面。未注射的一面可以用作控件。共同注射示踪剂（ 如 EGFP mRNA）以鉴定注射侧。在第二天，ea通过洗浴应用可以用药理试剂处理大量尾巴（〜st24）胚胎。心脏在第三天清晰可见。 DiI-AcLDL可以注射到心脏，注射后不久可以对血管进行成像。使用st33-37胚胎成像血管生长的动态或st42胚胎成像完全发育的血管。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55652/55652fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55652/55652fig2.jpg" /> 图2:在st42的DiI-AcLDL注射的位置。 42侧胚胎的侧面（ AB&#39; ）和腹侧（ CD ）视图。心脏在AC中的粉红色，从Xenbase（www.xenbase.org）拍摄的图像中。代表性的立体图像显示在A&#39;中 ， <strB， D 。对于注射，刺穿覆盖心脏的皮肤（ C ，实心箭头），进行线性切口，并横向打开皮肤以暴露心脏（红色箭头）。切开后，心脏将明显区分注射（ D&#39; ）。刻度棒=200μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55652/55652fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55652/55652fig3.jpg" /> 图3:预期结果（立体图像）。来自阶段37/38（ A ）和阶段42（ B ）胚胎的代表性荧光图像。显示胚胎的左侧。后基底静脉（PCV）尾静脉延伸，其中间质静脉（ISV）从背侧分支到尾部至尾部即在第37/38（ A ）阶段只有流行的ISVs可见，大多数ISV都是由阶段42（ B ）形成的。如果注射不足量的DiI-AcLDL，即使在30分钟（ C ）之后，PCV也不会清楚可见。在这种情况下，可以注入更多的DiI-AcLDL。如果DiI-AcLDL意外注射到其他器官（ D ）中，则丢弃胚胎。刻度棒= 500μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55652/55652fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" src="/files/ftp_upload/55652/55652fig4.jpg" /> 图4:Tie2信号对ISV发展的影响。 Tie2基因表达被翻译阻断反义吗啉代寡核苷酸（Tie2MO）的卵裂球注射敲低。对照吗啉（CoMO）具有相同的分子量，但是n靶向热带非洲爪蟾中的任何基因。共同注射mRNA编码的组成型活性Tie2突变体（caTie2）以拯救Tie2敲低表型。与对照（ A ）相反，Tie2MO注射导致ISV的长度和数量的显着降低（ B ）。这种表型被caTie2过度拯救，其显示具有旺盛的侧枝的ISV（ C ）。图5中用蓝色虚线框表示的区域中的ISV的复杂度被量化。比例尺=200μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55652/55652fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" src="/files/ftp_upload/55652/55652fig5.jpg" /> 图5:静脉复杂度的定量。 （ A ）ISV的复杂性可以由静脉复杂度指数（VCI）表示。（ BD ）VCI由图4所示的胚胎计算。比例尺=200μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55652/55652fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis at JoVE.com

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

该方案证明了一种基于荧光的方法来显示脉管系统并量化其在热带非洲爪蟾中的复杂性 。在遗传和/或药理学操作之后，将荧光染料注射到胚胎的跳动心脏中以在体内研究心血管发育后几分钟可以对血管进行成像。

胚胎血管生成的可视化和定量分析<em&gt;热带非洲爪蟾</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

visualization-quantitative-analysis-embryonic-angiogenesis-xenopus

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

8.3K 次观看.  Yonsei University College of Medicine. 该方案证明了一种基于荧光的方法来显示脉管系统并量化其在热带非洲爪蟾中的复杂性 。在遗传和/或药理学操作之后，将荧光染料注射到胚胎的跳动心脏中以在体内研究心血管发育后几分钟可以对血管进行成像。 

视频: Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

操作和<em&gt;在体外</em中&gt;成熟<em&gt;非洲爪蟾</em&gt;卵母细胞，其次是胞浆内单精子注射，研究胚胎发育

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

科研

发育生物学

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

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

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

在胚胎小鼠心脏采用免疫荧光心肌发展分析

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

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

通过在胚胎梅克尔软骨Zebrabow克隆细胞分析软骨插层和定向扩散的可视化

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

利用共聚焦分析<em&gt;非洲爪蟾</em&gt;调查的Wnt和Shh形态发生素梯度的调节剂

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

干细胞样<em&gt;爪</em&gt;胚胎外植体，研究早期神经发育特点<em&gt;体外</em&gt;和<em&gt;在体内</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

转录组分析]<em&gt;活体</em&gt;生产牛胚胎植入前胚胎采用双色微阵列平台

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Technique to Target Microinjection to the Developing Xenopus Kidney

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 embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

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 ...

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 ...

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

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

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

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

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