我们感谢李院长分享他的实验室的主动脉 SMC 隔离协议。资助由国立卫生研究院 (R21NS088854、R01HL125815 和 R01HL133016 博士 G)、美国心脏协会 (补助金14GRNT19990019 至 D.M.G.) 和耶鲁大学 (布朗-柯克斯奖学金提供给上午和启动资金 D.M.G.)。

所有鼠标协议均由耶鲁大学机构动物保育和使用委员会批准. 
 1. 在体内 胚胎命运图和克隆分析 注意: 我们在发展和疾病模型中广泛使用这些方法来评估细胞的起源及其克隆结构 7 , 8 , 9 , 10 . <ol> <li> 设置与 CreER 和小鼠之间的交配与一个综合招聘报告. 
	注: CreER 用于 SMC 标记;Myh11-CreERT2 或 Acta2-CreERT2 鼠标 11 , 12 通常用于此目的。我们使用了 ROSA26R-CreERT2 小鼠 13 来广泛标记胚胎组织。
	<ol> <li> 用于克隆分析, 请使用多色综合招聘报告员 ( 例如, 五彩纸屑或彩虹 [Rb] 鼠标) 8 , 14 , 15 和 "命运映射", 使用单色 ( 例如, ROSA26R-YFP 鼠标 16 ) 或色的记者 ROSA26R-mTomato-mGFP (mTmG) 17 . 
		注: 此繁育方案使用 2-6 月大的成年小鼠 (一般为30克), 并将在 CreER 和一名综合招聘报告中生成胚胎. </li>
	</ol> </li> <li> 使用金属探头在早上检查阴道插头, 并考虑在插头检测当天中午为胚胎日 (E) 0.5. </li>
	当检测到插头时, <li> 将雌性与男性分开. </li>
	<li> 准备 4-OH-他莫昔芬和他莫昔芬工作解决方案。
	<ol> <li> 为 4-OH-他莫昔芬, 溶解5毫克到50和 #181; l 100% 乙醇, 涡流为2分钟, 然后添加450和 #181; 玉米油的 l。在冰, 几种在输出水平2为十年代周期与休息在周期之间直到样品冷却下来。样品完全溶解后 (通常为 4-5 循环的超声), 采取200和 #181; l 声溶液, 溶解于1800和 #181; l 玉米油, 以使最终的工作解决方案 (4-OH-他莫昔芬, 1 毫克/毫升). </li>
		<li> 对于他莫昔芬, 在100% 乙醇和涡流中稀释到50毫克/毫升, 直到溶解为止。在玉米油中稀释, 使最终的工作溶液 (三苯氧胺, 10 毫克/毫升) 和搅拌45和 #176; C, 以确保它完全溶解. </li>
	</ol> </li> <li> 早期胚胎诱导, 注入 4-OH-他莫昔芬腹腔到一个怀孕的大坝。
	<ol> <li> 使用 4-OH-他莫昔芬注射液 (高达〜150和 #181; g 每怀孕的老鼠, 或大约5毫克/千克体重) 在 E5.5 和此后, 因为他们产生可行的大坝, 胚胎, 和幼崽. </li>
		<li> 用他莫昔芬在 0.5-1.5 毫克 (或 17-50 毫克/千克) 的情况下, 在 E9 和此后的胚胎中使用的水坝。使用30G 针进行所有注射. 
		#160; 注: 通过0.22 和 #181 过滤; m 过滤器通常用于在注射前对所有混合物进行消毒。和 #160; </li> </ol> </li> <li> 用于克隆分析, 使用高剂量的腹腔注射 4-OH-他莫昔芬 (, 5 毫克/千克) 或他莫昔芬 (, 50 毫克/千克) 来标记多个细胞。
	<ol> <li> 要标记单个单元格, 请滴定 4-OH-他莫昔芬或他莫昔芬的剂量, 这样, 在感兴趣的组织 ( 即, 主动脉) 中, 几乎所有的胚胎都被分析 (如下所述, 在本节末尾) 没有细胞标记或只有细胞单一颜色的;这是阈值剂量. </li>
	</ol> </li> <li> 在中的妊娠期内标记细胞, 并追踪他们的命运或克隆在产后小鼠, 在怀孕的大坝的腹腔内注射孕酮伴随与他莫昔芬在 1:2 (孕酮: 他莫昔芬)剂量比. 
	注: 他莫昔芬的剂量是 17-50 毫克/千克和孕酮剂量是 8.5-25 毫克/千克 ( 即 , 他莫昔芬剂量的一半)。孕酮注射液可延迟分娩 1-2 天. </li>
	<li> 安乐用一个开放滴法将大坝孕育在一个理想年龄的胚胎, 在那里, 大坝被放置在一个装有棉花或纱布的容器中, 用未稀释的异氟醚浸泡。通过打开胸腔以诱发气胸、切除重要器官和/或执行颈椎脱位确认死亡. </li>
	<li> 用70% 乙醇清洗腹部。用剪刀切开腹部, 解剖子宫。从子宫中取出胚胎, 用钳子将胚胎从胎盘和卵黄囊中仔细分离. </li>
	<li> 安乐 E15 或更年长的胚胎通过手术剪刀或锋利的刀片进行颈椎脱位或斩首. 
	注: 小于 E15 的胚胎在母亲的安乐死和/或从母体中取出胚胎后迅速死亡. </li>
	<li> 将胚胎放在 ice-cold PBS 中, 用剪刀将一小片尾巴切开, CreER 和综合招聘报告员. </li>
	<li> 将4% 甲醛中的胚胎固定在4和 #176; C 为2小时. 在 pbs 中冲洗胚胎三次, 然后在30% 毫升的 pbs 中用15蔗糖孵育胚胎, 等待直到它们下沉, 这可能需要两天时间. </li>
	<li> 用最佳切削温度 (OCT) 化合物填充塑料冷冻模具, 最高可达到2/3 的最大水平。将每个胚垂直放置在模具中, 完全浸没在 OCT 中. 室温下孵育10分钟以最小化气泡. </li>
	<li> 将冰冻组织块直立在干冰中, 70% 乙醇浴冻结。将块存储在-80 和 #176; C. </li>
	<li> 将低温温度设置为-22 和 #176; C 和切口块通过整个主动脉、10-20 和 #181 来产生横截面; m 厚。在室温下将这些幻灯片在-80 和 #176 之前干燥30分钟; C. </li>
	<li> 在 RT 中对幻灯片进行解冻, 避免光的荧光。用 PBS-Tween20 0.1% 冲洗幻灯片。
	<ol> <li> 进行克隆分析, 染色的幻灯片核 (DAPI, 5 毫克/毫升) 和直接图像其他荧光在彩虹综合招聘记者 (蔚蓝: 433 nm 激发最大, 475 nm 的排放量最大; mOrange: 548 nm, 562 nm; mCherry: 587 nm, 610 nm)。
		<ol> <li> 使用以下过滤器来检测荧光与直立荧光显微镜: DAPI (励磁最大/带宽 350/50 nm, 发射最大/带宽 460/50 nm), 蔚蓝 (励磁436/20 毫微米, 放射480/40 毫微米), 得克萨斯红色 (励磁 560/40毫微米, 发射630/75 毫微米) 和自定义过滤器分离 mCherry 从 mOrange (励磁577/10 毫微米, 放射630/50 毫微米). </li>
		</ol> </li> <li> 用于与 mTmG 的命运映射报告, 通过直接成像或使用免疫检测 GFP (484 nm, 510 nm)。为成像用直立的萤光显微镜使用 GFP 过滤器 (励磁470/40 毫微米, 放射525/50 毫微米). </li>
		<li> 对于免疫, 在4和 #176 的一夜之间孵育具有主抗体的切片, 在 PBS 中用 0.1% X-100, 然后用二次抗体孵育 1 h. 使用 anti-CD31 (最终浓度0.0016 毫克/毫升), 抗 GFP (0.006 毫克/毫升) 和Cy3-directly 共轭抗 SMA (1:500 最终稀释) 主要抗体和使用继 antibodies 直接共轭到荧光 (1:500 最后稀释) (请参见 材料表 ). </li>
	</ol> </li> <li> 垂直荧光显微镜 (放大倍数: 4X-20X) 或共聚焦显微镜 (10X-63X) 的图像幻灯片。对于高放大率, 使用共聚焦成像63x 油目标, 数值孔径为 1.4-0.6, 帧大小为 1024 x 1024 像素. </li>
</ol> 2。外植体胚胎主动脉培养 注意: 此方法以前用于评估整合素 beta3 在说明的狭窄中的作用 民族解放军 (-/) 胚胎主动脉 9 , 18 . <ol> <li> 安乐在 E15.5 上由过量的异氟醚吸入引起的定时怀孕的大坝, 如步骤1.8 所示. </li>
	根据以上步骤 1.9-1.10, <li> 收获和安乐胚胎. </li>
	<li> 将胚胎仰并将伸展的四肢固定在解剖板上。可视化解剖立体和使用剪刀切割从腹部垂直切口通过胸骨的上部胸部。切除胸腺、气管、肺部、食道、肝脏和肠道. </li>
	<li> 用镊子轻轻拉心腹, 用剪刀解剖主动脉, 远离胸腔和腹腔的背侧。释放主动脉, 在近端根部和远端腹部位置切开. </li>
	<li> 在细胞培养罩中将主动脉置于 ice-cold 无菌 PBS 中。转移主动脉到一个24井板和文化它在 DMEM 与 0.5% FBS 为 24 h 在37和 #176; C. 
	注: 培养基可辅以阻断抗体 ( 例如, anti-integrin 和 #945; v 和 #946; 3 抗体 at 0.02 毫克/毫升 9 ; 查看 材料表 ) 或药理剂. </li>
	<li> 用 PBS 清洗主动脉两次, 然后用4% 甲醛20分钟来修复它. </li>
	<li> 用 pbs 冲洗三次, 并将主动脉转移到1.5 毫升的导管, 在 pbs 中使用30% 蔗糖。主动脉下沉后, 按照上面的步骤 1.13-1.15 所述, 制作冰冻组织块、切片和免疫. </li>
</ol> 3。SMC 与围产期主动脉的分离 注意: 该方法目前正被用于比较来自 民族解放军 (-) 和野生围产期小鼠的主动脉校董的生物学. 
<ol> <li> 安乐在出生后的一天 (P) 0.5, 如上文步骤1.10 所述, 由颈椎脱位或用手术剪刀或锋利的刀片斩首. </li>
	<li> 执行步骤 2.3, 然后在打开胸腔和腹部后, 用23G 针穿刺左心室。使用塑料管将针头与无菌的 pbs 注射器相连, 垂直放置在高架位置, 以便 pbs 以重力的状态流入左心室。允许将无菌 PBS 注入左心室, 直到肝脏 blanches。
	<ol> <li> 使用镊子去除主动脉外侧的外组织, 并按照步骤2.4 中的描述对主动脉进行解剖. </li>
	</ol> </li> <li> 在 DMEM (500 和 #181; L) 的单个解决方案中, 使用 225 u/毫升胶原酶, 2.25 u/毫升弹性蛋白酶, 和1x 抗生素-抗, 在37和 #176 中进行45分钟的补充; c. 手动摇动管每 5-10 min. </li>
	<li> 在无菌细胞培养罩中, 通过移200和 #181, 将经消化的组织 titurate; L 吸管生成单细胞悬浮液。将单细胞悬浮液转移到15毫升的管中, 加入2毫升的 DMEM, 并在 920 x g 离心5分钟, 在25和 #176; C. </li>
	<li> 丢弃上清。重3毫升的 SMC 培养基 (DMEM 10% FBS 补充1和 #181; g/毫升 rhFGF, 10 和 #181; g/毫升肽, 100 U/毫升青霉素/链霉素, 和2.5 和 #181; g/毫升两性霉素 B)。转移到一个35毫米的文化板块. 
	注意: 在最初的隔离, 〜40万细胞是从一个单一的 P0.5 野生主动脉;这些细胞中有30万是校董的. </li>
	<li> 将单元格培养为37和 #176; C 每3天更换为新鲜的 SMC 培养基。使用标准技术与胰蛋白酶, 通过细胞时汇合。对于实验, 使用 3-7 通道中的单元格. </li>
</ol> 4。皮下渗透微型泵在妊娠 (或非怀孕) 小鼠中的放置 注意: 此方法以前用于持续提供药理剂 (如: "整合素和 #946; 3 和 #946; 5 抑制剂cilengitide) 到胚胎 在子宫内 9 。泵入到怀孕的水坝在 E13 . 5 和保持直到分娩 . 
<ol> <li> 麻醉 2-5% 异氟醚在氧气中的小鼠 (流量为1升/分) 用于诱导和1-3% 的维护。使用脚趾捏反射来监测麻醉的水平, 并根据需要调整麻醉剂。管理皮下丁丙诺啡 (0.05-0.1 毫克/千克) 镇痛. </li>
	<li> 用快船刮下背部。在手术中使用无菌窗帘、手套和器械, 并在加热手术盘上维护小鼠. </li>
	<li> 将每只老鼠放在俯卧位置, 用控告后再用异丙醇擦洗背部。大约 3-5 cm 侧到尾巴的底部在下背部, 使用手术刀, 使水平皮肤切口 0.5-1.0 厘米的长度, 而不伤害部下的肌肉. </li>
	<li> 插入一个止血到切口, 并创建一个皮下口袋 rostrally 泵植入通过开放和关闭止血颌骨. </li>
	<li> 根据制造商和 #39 的指导原则 (请参阅 材料表 ), 以选择的代理填充渗透 mini-pump。将填充泵插入口袋, 然后用夹子或6-0 吸收缝合线关闭切口。确保手术总时间少于10分钟. </li>
	<li> 手术后, 监测小鼠每15分钟, 直到他们已经从胸骨卧床恢复。管理皮下丁丙诺啡 (0.05-0.1 毫克/千克) 每 6-12 小时为 48 h。当老鼠完全从全身麻醉中恢复过来时, 每天都要监测它们。删除剪辑或缝合线 7-10 天后. </li>
</ol>

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Defining a mesenchymal progenitor niche at single-cell resolution.</a> Science. 346, 1258810 (2014).</li><li>Srinivas, 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=Cre+reporter+strains+produced+by+targeted+insertion+of+EYFP+and+ECFP+into+the+ROSA26+locus.">Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.</a> BMC Dev Biol. 1, 4 (2001).</li><li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li><li>Li, D. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elastin+gene+is+disrupted+by+a+translocation+associated+with+supravalvular+aortic+stenosis.">The elastin gene is disrupted by a translocation associated with supravalvular aortic stenosis.</a> Cell. 73, 159-168 (1993).</li><li>Li, D. Y., 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=Novel+arterial+pathology+in+mice+and+humans+hemizygous+for+elastin.">Novel arterial pathology in mice and humans hemizygous for elastin.</a> J Clin Invest. 102, 1783-1787 (1998).</li><li>Li, D. Y., 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=Elastin+point+mutations+cause+an+obstructive+vascular+disease,+supravalvular+aortic+stenosis.">Elastin point mutations cause an obstructive vascular disease, supravalvular aortic stenosis.</a> Hum Mol Genet. 6, 1021-1028 (1997).</li><li>Pober, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Williams-Beuren+syndrome.">Williams-Beuren syndrome.</a> N Engl J Med. 362, 239-252 (2010).</li><li>Pober, B. 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作者没有什么可透露的。

与大鼠主动脉及其主要分支在成人病理条件下, 如动脉粥样硬化模型的广泛研究不同, 对胚胎和围生期主动脉的形态发生和发病机制的了解较少。在这里, 我们专注于胚胎/围产期主动脉, 特别是校董, 并提供协议, 以研究主动脉通过在体内, 组织外植体, 和 SMC 隔离方法。这些免费的方法为研究者提供了不同的方法来研究胚胎/围产期主动脉。
克隆分析是一种非常强大的方法...

多细胞生物体的循环系统功能向不与外界环境接触的细胞输送养分和氧气, 并从这些细胞中去除废物和二氧化碳。在脊椎动物中, 初级循环系统由心脏组成, 它通过一系列血管泵血。大血管的壁, 如动脉和静脉, 由三层组成: i) 内皮细胞内膜, 或内部层;2) 培养基, 或中间层交替圆周细长平滑肌细胞校董和弹性片状;和 iii) 的膜, 或外层结缔组织和成纤维细胞。绝大多数的血管生物学研究集中在内皮细胞, 通过血管生成研究新的内皮细胞内衬管的形成。相比之下, 校董得到的关注相对较少。然而, 校董是一个关键的细胞类型, 在建设正常动脉壁和血管病理。
主动脉是体内最大口径的动脉, 接受心脏左心室的输出。它受到各种人类疾病的折磨, 包括动脉粥样硬化、动脉瘤和解剖。在成人机体中, 主动脉及其主要分支在血管疾病模型中得到了深入的研究。例如, 高脂肪饮食喂养小鼠是无效的基因编码低密度脂蛋白受体或载脂蛋白 E, 发展动脉粥样硬化, 和最近的命运测绘研究表明, 预先存在的校董产生多种细胞类型的动脉粥样硬化斑块1。在主动脉瘤中, 病理改变包括 SMC 细胞凋亡和细胞外基质重塑2,3。
在胚胎和围产期期间, 对 SMC 的形态发生和发病机制的认识显著减少。在这里, 我们提供了研究胚胎和围产期主动脉校董在体内, 组织外植体和孤立细胞的协议。例如, 该协议的第一部分揭示了胚胎小鼠的命运映射和克隆分析。重组在细胞特异启动子的控制下表达, 促进特定细胞及其子代的标记4,5,6;然而, 在小鼠胚胎发育过程中, 对细胞特异标记的时间控制可能具有挑战性。在这方面, 在校董 (例如, Myh11或Acta2) 和一个综合招聘报告员的 CreER 下, 我们提供了在怀孕的水坝中注射他莫昔芬或其活性代谢物 4-OH-苯氧胺的方法。用于分析胚胎或产后后代的标记细胞。此外, 与命运测绘研究, 这是以一个单一的记者荧光1,7, 克隆分析大大加强与多色综合招聘记者。
第二和第三部分的协议描述的方法, 分离和培养胚胎主动脉外植体和主动脉校董的新生儿, 分别。这些方法允许操纵信号通路, 特别是在主动脉外植体或校董, 并分析药物的直接作用。因此, 在兴趣组织中的特定基因的作用可以比传统的小鼠遗传操作更快速地进行筛选。此外, 孤立的 SMC 研究有助于分析细胞迁移和黏附, 这是技术上有限的在体内。
最后, 第四议定书部分划定了在怀孕 (或非) 小鼠体内装有药理剂的皮下渗透 mini-pump 的位置。这种方法有助于分析由于新陈代谢快而需要连续输液的药剂对胚胎发育的影响。经常注射的替代品对许多药物来说是不可行的, 应该避免, 因为它可能会在怀孕的大坝中造成严重的不适。

<table width="1061">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Tamoxifen</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:111px;">T5648</td>
			<td style="width:412px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Corn oil</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:111px;">C-8267</td>
			<td>Vehicle for tamoxifen</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">4-OH-tamoxifen</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:111px;">H7904</td>
			<td>Active metabolite of tamoxifen</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Progesterone</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:111px;">P8783-5G</td>
			<td>Use at half the concentration of tamoxifen</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">OCT compound</td>
			<td style="width:216px;">Sakura tissue tek</td>
			<td style="width:111px;">4583</td>
			<td>For making cryoblocks</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Cryomolds</td>
			<td style="width:216px;">Polysciences inc</td>
			<td style="width:111px;">18986</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">DAPI</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:111px;">D9542</td>
			<td>IHC staining of nucleus, final concentration 5 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy3 directly conjugated anti-SMA antibody</td>
			<td>Sigma</td>
			<td>A2547</td>
			<td>IHC staining of SMA, final dilution 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Anti-CD31 antibody</td>
			<td style="width:216px;">BD Pharmingen</td>
			<td style="width:111px;">550274</td>
			<td>IHC staining of GFP, final concentration 0.006 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Anti-GFP antibody</td>
			<td style="width:216px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">A-11121</td>
			<td>IHC staining of CD31, final concentration 0.0016 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibody goat anti-rabbit, Alexa 647</td>
			<td style="width:216px;">Life Technologies</td>
			<td>a21244</td>
			<td>IHC staining, final concentration 0.004 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibody goat anti-rabbit, Alexa 488</td>
			<td style="width:216px;">Life Technologies</td>
			<td>a11008</td>
			<td>IHC staining, final concentration 0.004 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">DMEM</td>
			<td style="width:216px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">10567-014</td>
			<td>For cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">FBS</td>
			<td style="width:216px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">10437028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Anti-integrin beta3 blocking antibody</td>
			<td style="width:216px;">BD Biosciences</td>
			<td style="width:111px;">553343</td>
			<td>Clone 2C9.G2, final concentration 0.02 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase</td>
			<td>Worthington Biochemical Corp</td>
			<td>44H14977A</td>
			<td>For digesting aorta</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elastase</td>
			<td>Worthington Biochemical Corp</td>
			<td>34K15139</td>
			<td>For digesting aorta</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Antibiotic-antimycotic (100X)</td>
			<td style="width:216px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">15240062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Recombinant human FGF</td>
			<td style="width:216px;">Promega</td>
			<td style="width:111px;">G5071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Recombinant human EGF</td>
			<td style="width:216px;">Promega</td>
			<td style="width:111px;">G5021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Penicillin/streptomycin (10,000 U/ml)</td>
			<td style="width:216px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Amphotericin B</td>
			<td style="width:216px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">15290026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Tissue culture plates</td>
			<td style="width:216px;">Corning</td>
			<td style="width:111px;">CLS430165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Alzet osmotic mini-pump</td>
			<td style="width:216px;">Durect Corporation</td>
			<td style="width:111px;">2001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECLIPSE 80i Upright Fluorescent Microscope</td>
			<td style="width:216px;">Nikon </td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"> TCS SP5</td>
			<td style="width:216px;">Leica</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Branson Sonifier 450</td>
			<td style="width:216px;">VWR</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Myh11-CreERT2 mice</td>
			<td style="width:216px;">The Jackson Laboratory </td>
			<td>19079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acta2-CreERT2 mice</td>
			<td style="width:216px;"></td>
			<td style="width:111px;"></td>
			<td>Obtained from lab of Dr. Pierre Chambon and Daniel Metzger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROSA26R-CreERT2 mice</td>
			<td style="width:216px;">The Jackson Laboratory </td>
			<td style="width:111px;">8463</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROSA26R(mTmG/mTmG) mice</td>
			<td style="width:216px;">The Jackson Laboratory </td>
			<td> 026862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROSA26R(EYFP/EYFP) mice</td>
			<td style="width:216px;">The Jackson Laboratory </td>
			<td> 006148 </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROSA26R(Confetti/Confetti) mice</td>
			<td style="width:216px;">The Jackson Laboratory </td>
			<td>13731</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROSA26R(Rb/Rb) mice</td>
			<td style="width:216px;">Lab of Dr. Irv Weissman</td>
			<td style="width:111px;"></td>
			<td style="width:412px;">Obtained from lab of Dr. Irv Weissman</td>
		</tr>
	</tbody>
</table>

using in vivo and tissue and cell explant approaches to study the morphogenesis and pathogenesis of the embryonic and perinatal aorta

主动脉是体内最大的动脉。主动脉壁由内皮细胞内层、交变弹性板中层和平滑肌细胞 (校董)、外层成纤维细胞和胞外基质组成。与广泛研究的病理模型 (例如,动脉粥样硬化) 在成人主动脉, 更少的是已知的胚胎和围产期主动脉。在这里, 我们专注于校董, 并提供协议, 分析胚胎和围产期主动脉校董在正常发育和疾病的发生和发病机制。具体来说, 包括的四项协议是: i)在体内胚胎的命运映射和克隆分析;二) 外植体胚胎主动脉培养;三) SMC 与围产期主动脉隔绝;和 iv) 皮下渗透 mini-pump 放置在怀孕 (或非) 小鼠。因此, 这些方法有助于调查的起源 (s), 命运, 和克隆结构的校董在主动脉在体内。他们允许通过持续接触药理学药物来调节胚胎主动脉形态的在子宫内。此外, 孤立的主动脉组织外植体或主动脉校董可用于在诸如 muscularization、增殖和迁移等基本过程中获得对特定基因靶点作用的洞察。这些假说生成实验的孤立校董和说明主动脉, 然后可以评估在体内的情况下, 通过药理和遗传方法。

在一个代表性的克隆分析校董的胚胎突变为民族解放军(基因编码细胞外基质蛋白弹性),民族解放军(+/-), Acta2-CreERT2 小鼠交配的民族解放军(+/-)鼠也携带多色 ROSA26R(rb/rb) 记者。如步骤1所述, 插头被检查, 怀孕的水坝是诱发了一个单一的他莫昔芬注射液 (1.5 毫克) 在 E12.5, 他们牺牲在 E18.5。胚胎被收割、固定、冷冻和基因。?...

Watch this Scientific Journal Video about Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta at JoVE.com

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

使用体内和组织和细胞外植体方法研究胚胎和围产期主动脉的形态和发病机制

用于研究胚胎和围产期小鼠主动脉的协议使用在体内克隆分析和命运图, 主动脉外植体和孤立平滑肌细胞在这里详述。这些不同的方法有助于调查胚胎和围产期主动脉在正常发育和疾病的发病机制。

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

using-vivo-tissue-cell-explant-approaches-to-study-morphogenesis

Research

JoVE Journal

Developmental Biology

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

8.4K 次观看.  Yale University School of Medicine. 用于研究胚胎和围产期小鼠主动脉的协议使用在体内克隆分析和命运图, 主动脉外植体和孤立平滑肌细胞在这里详述。这些不同的方法有助于调查胚胎和围产期主动脉在正常发育和疾病的发病机制。

视频: Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

科研

发育生物学

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, ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

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

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

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 ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

卵巢组织培养对小鼠卵巢现象的可视化

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

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 ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

晚期胚胎 果蝇 性腺的解剖和活体成像

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

研究脊椎动物轴向伸长和分割的三维和四维可视化分析方法

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

使用浸泡珠测定分析鸡胚胎发生过程中的细胞分化，形态发生和图案化

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...