一个单细胞消融从甲壳动物端足八细胞胚胎<em> Parhyale hawaiensis</em
Summary
的端足Parhyale hawaiensis是一种很有前途的模式生物的甲壳类动物胚胎学和比较节肢动物的发展和演变的研究。本协议描述为从Parhyale的早期卵裂期胚胎手工清除单卵裂球的方法。
Abstract
的端足Parhyale hawaiensis是在世界范围内潮间带海洋栖息地发现了一个小的甲壳类动物。在过去的十年中，Parhyale已经成为一种很有前途的模式生物发展的实验室研究，提供了一个有用的外类群相比，以及节肢动物的研究模式生物果蝇 。而相比之下， 果蝇合胞分裂，Parhyale的早期分裂是holoblastic。使用注入早期卵裂球示踪染料命运映射表明，所有三种胚层和中胚系由8细胞阶段建立。在此阶段，3卵裂球注定要产生外胚层，三是注定要产生的中胚层和剩余的两个卵裂球为内胚层和胚系的前体分别。然而，卵裂球烧蚀实验表明，Parhyale胚胎也具有显著的监管capabilitiES，使得卵裂球烧蚀在8 – 细胞期的命运可以接管的一些剩余的卵裂球的后代。卵裂球消融以前已经描述的两种方法之一：注入和光毒性的染料或手工切除的后续激活。然而，光烧蚀杀死卵裂球，但不从胚胎去除死细胞体。因此，完整的物理去除特定的卵裂球可能是消融的某些应用场合的首选方法。在这里，我们提出了一个协议，用于从Parhyale胚胎八细胞阶段手工清除单个卵裂球，说明在保持其余卵裂球存活完好完全去除细胞体所必需的工具和手动程序。这个协议可以被应用到任何Parhyale细胞在8细胞阶段，或以其他早期卵裂阶段的卵裂球。此外，原则上这个协议可以适用于早期CLEA其他holoblastically裂解海洋无脊椎动物vage阶段的胚胎。
Introduction
在片脚甲壳类Parhyale hawaiensis已经出现在过去的十年是一个很有前途的模式生物与进化发育生物学研究1利用潜力巨大。间的节肢动物，大多数的模型系统是昆虫，和最广泛研究的是这些果蝇黑腹果蝇。D.果蝇是双翅目昆虫的一员，并作为衍生对于那些基部分枝昆虫2这样的陈列着许多胚胎学特征。此外，嵌套虫亚门Pancrustacea 3之内，这意味着昆虫有长期存在的“自然”组名为pancrustaceans内自己最亲近的亲戚，而这个群体是并系。这表明，除了基部分枝昆虫模型，其他甲壳类动物的研究来获得发展特征的进化史的一个更广阔的视野一已经这么好研究D. ND分子机制果蝇 。然而，很少甲壳类动物已经确立为发展试验实验室分析。的端足P. hawaiensis是高度易处理的实验室模型系统，适合于一系列的实验技术。端足目动物显示了许多独特的功能，他们的父母超目Peracarida（沙滩料斗，飞毛腿，和良好虾）内，并因此认为是相对这个群体的甲壳类中派生的。然而，相对容易通过Parhyale提供胚胎和功能基因操作使这个端足一个有价值的除了模式生物的当前库存。
作为实验室动物， 体育hawaiensis提供了许多优势。动物能忍受广泛的温度范围内和盐度，并在人工海水中1的大文化生存良好。这是很容易分辨本月与基于明确的形态差异，最突出的是大的，大呼过瘾，躯干前部的附肢，男性使用交配过程中掌握女性EEN男性和女性。对于胚胎学和发育的工作，P. hawaiensis有几个非常吸引人的功能。胚胎发生持续约10天的时间，性成熟大约六个星期在28ºC（但要注意，Parhyale生存以及在温度范围从大约20-30ºC，并且详细的发育分期信息可用于在18ºC 4提高胚胎，25ºC 4，和26ºC 5,6）。成人交配常年在实验室里，所以胚胎可在一年中的任何时间。雌虫2-20（取决于年龄的女性）受精卵到腹侧育雏囊位于所述第一多对腿部（ 图1A和1B）之间，并且可以收集这些胚胎S在发育早期没有杀害女性或损坏胚胎（ 图1C）。胚胎在人工过滤的海水中生存，通过孵化，可以固定为后续基因表达或组织学分析7，并有详细的临时表可以精确识别的进步，通过发展5。健壮的协议已被用于原位杂交8-15或免疫染色4,16,17进行基因表达分析，通过RNA干扰13,15或吗啉12，稳定的种系功能性敲除转基因18。使用转基因系统中，诱导表达14和增强子陷阱19的方法也可以被用来研究在巴斯德基因功能hawaiensis。而一个公开的基因组序列是当前不可用，含成绩单转录和卵子胚胎发育过程中产生的蜜蜂Ñ 从头组装和注释20，并存放于一个可搜索的数据库21，促进基因的发现。总之， 体育hawaiensis适用于多种实验和遗传方法来了解发展的高度听话的模式生物。
不同于D的早期合体分裂黑腹果蝇，P. hawaiensis胚胎裂解holoblastically以下受精（ 图2A）。谱系追踪分析表明，由第三切割，每个所述第三切割卵裂球的具体注定要产生1的三个胚层或中胚系6（ 图2B）。这些数据，连同微阵列数据22，细胞谱系分析6,23，和卵裂球分离实验4表明，发展潜力被隔离至少某些第三裂解卵裂球由CEL的不对称继承升命运的决定因素。因此，在该种系的前体（在Parhyale细胞谱系命名法6称为“ 克 ”）除去在8细胞期卵裂球消融的实验中，胚胎缺乏生殖细胞发育后期4，由于缺乏细胞所指示的表达该蛋白瓦萨，这是一个种系的标记中最后生动物24。与此相反，体细胞卵裂球消融的实验表明，P. hawaiensis胚胎也具有显著的监管能力，使得消融在八细胞阶段的中胚层或外胚层前体卵裂球的命运可以接管一些剩余的卵裂球25的后裔。如何调控细胞命运的更换可能发生，自治区细胞命运采用体细胞卵裂球的情况下，仍然不得而知。如卵裂球烧蚀实验胚胎学技术可以在understan是有用的鼎的相对自主性和细胞命运的决定非自治26,27，因此是在P的学习兴趣hawaiensis胚胎。
在这种证明调控更换外胚层和中胚层谱系的实验，卵裂球消融注射28和光毒性染料25的后续激励执行。虽然这种技术是在杀死注射卵裂球（次）有效，但并没有完全从胚胎中去除死细胞体。此外，差异已通过注射卵裂球用荧光示踪剂谱系28,29收集到胚胎的原肠胚形成期细胞谱系的数据，以及通过相同的胚胎阶段23的发展如下沉着卵裂球收集到的数据之间的变化。因此，完整的物理去除特定的卵裂球可能是消融的某些应用场合的首选方法。
我们以前出版的细胞谱系分析的结果胚胎中，单个细胞进行人工消融23。然而，从早期卵裂期胚胎取出单个卵裂球所需的细腻操作尚未充分说明。在这里，我们提出了一个协议的集合P的hawaiensis胚胎和从一个八细胞期胚胎的单个卵裂球的手册消融。该方法的目标是实现从胚胎完全除去细胞体的，允许观察细胞行为和胚胎发生和胚胎后发育过程中的剩余细胞的细胞命运的能力。我们的协议表示除去生殖系前体克 （ 图2C），但可以应用到任何细胞在8细胞阶段，或者早期卵裂阶段的卵裂球。原则上，该协议可以适用于从行吟诗人早期卵裂期胚胎取出单个细胞řholoblastically裂解海洋无脊椎动物。
Protocol
Representative Results
Discussion
我们描述了一个协议，用于手动消融和从端足P的早期卵裂阶段的完整的物理去除单个卵裂球hawaiensis。我们证明使用该协议的通过从八细胞阶段的胚胎取出单个生殖系前体细胞克 ，并表明消融已成功通过确认没有G'女儿细胞后的胚胎发育。这个协议可以被用来从胚胎早期卵裂阶段除去任何micromeres的。使用该协议，以除去macromeres在此阶段，或卵裂球在第一?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Rayhan阿里夫和Hassaan Shahawy相机工作，Tripti Gupta和Frederike Alwes寻求协助炼细胞消融技术，Extavour实验室成员上的数据，视频和手稿的反馈。这项工作是部分由哈佛干细胞研究所的支持（种子格兰特编号SG-0057-10-00）埃利森医学基金会（新学者奖数量AG-NS-07010-10），以专家咨询小组，并以哈佛学院研究计划奖ARN。
Materials
| 15 cm Pasteur pipette | Wheaton | 53499-630 | For transferring intact and blastomere-ablated embryos from dish to dish. VWR |
| 22 cm Pasteur pipette | Wheaton | 53499-632 | For making mouth pipette tips. VWR |
| 3 cm petri dishes | Thermo Scientific | 25382-334 | Use some unaltered to collect and store embryos; coat some with a layer of Sylgard (see below) to create a working surface for blastomere ablations. VWR |
| 48-well plates | Greiner Bio-One | 82051-004 | For culturing embryos following ablation. VWR |
| Bottle top filters 0.2 micron | Nalge Nunc International | 28199-296 | For creating FASW (See below) VWR |
| Bunsen burner | VWR | 89038-530 | For creating tungsten wire tool and fine tip of mouth pipette. VWR |
| Diamond scribe | Musco Sports Lighting | 52865-005 | For creating wide-mouthed Pasteur pipettes for collecting couples. VWR |
| Forceps | Fine Science Tools | 11050-10 | For holding anaesthetized females during removal of embryos from brood pouch. These do not have to be fine-tipped Dumont forceps – the tips can be at least as blunt as that in the forceps for which the catalogue number is listed here. Fine Science Tools |
| Fungizone-Amphotericin B | Sigma | A2942 | Add to FASW to a final concentration of 1mg/ml, and use this solution to culture blastomere-ablated embryos. Sigma Aldrich |
| Glass capillaries 4 inches long, 1/0.58 OD/ID (mm) | World Precision Instruments | 1B100-4 | need to include glass capillary and needle puller machine? World Precision Instruments |
| Glass watchglass 300 mm diameter | Electron Microscopy Sciences | 70543-30 | For use in removing embryos from the brood pouch of anaesthetized females. Electron Microscopy Sciences |
| Instant Ocean Artificial Sea Water (ASW) | Instant Ocean | IS160 | Make ASW by dissolving salt in deionized water to a salinity of X. Aquatic EcoSystems |
| Instant Ocean Filtered Artificial Sea Water (FASW) | Instant Ocean | IS160 | Use 0.2 micron filters to sterilize ASW. Make up in small amounts (< 250 ml) as needed. Aquatic EcoSystems |
| Kimwipes | Kimberly-Clark | 21905-026 | For safely breaking off the fine end of Pasteur pipettes modified for collecting couples. VWR |
| Mouth pipette adaptor | Drummond Labware | A5177-5EA | Insert the fine pulled pasteur pipette tip into this end, to be used for removing extruded cell contents during the ablation procedure. Sigma Aldrich |
| Mouth pipette tubing | Aldrich | Z280356 | Cut this to be long enough to comfortably hang around your neck during the ablation procedure. Sigma Aldrich |
| Needle Puller | Sutter Instrument Company | P97 | For making needles from glass capillaries for puncturing the blastomere to be ablated. Sutter Instrument Company |
| Penicillin-Streptomycin Solution | Mediatech | 45000-650 | Add to FASW to a final concentration of 100 units/ml penicillin and 100 mg/ml streptomycin, and use this solution to culture blastomere-ablated embryos. VWR |
| Rubber bulb | Electron Microscopy Sciences | 100488-418 | For using Pasteur pipettes to transfer embryos from dish to dish. VWR |
| Sylgard 184 | K. R. Anderson, Inc. | NC9659604 | Make up according to manufacturer's instructions. Pour a layer 2-5 mm thick into a 3cm petri dish to create a working surface for blastomere ablations. Fisher Scientific |
| Tungsten wire 0.004 inches diameter | A-M Systems | 719000 | Use this to make a tool for removing embryos from the brood pouch of anaesthetized females. A-M Systems |
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