新颖的指标来表征线虫胚胎伸长率<em>秀丽隐杆线虫</em
概要
Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.
Abstract
Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.
Introduction
近50年来的线虫确立了自己作为一个强大的模型来研究发展，神经生物学，进化，宿主-病原体相互作用等重要问题1这种模式在发展研究的优势在于：它的生命周期短的3天;与这些动物可以遗传改造的容易性;其透明度，使细胞的位移和形态的观察活体动物，其发展的主要是多余的子宫。线虫的发育阶段涉及胚胎和四个幼虫期（L1到L4），其次是成年。在胚胎发育期间，表皮形态吸引了相当多的关注其能更好地理解如何上皮细胞迁移作为一个群体，他们是如何重新组织他们的路口和修改其个人的形态和功能性上皮细胞内的相对定位能力。表皮形态被分为四个阶段：背侧嵌入由背表皮细胞的重组，称为皮下组织;腹侧外壳，包括在朝向腹侧中线从而包围在上皮细胞单层胚胎腹侧皮下细胞的迁移;早期和晚期延伸改造豆形的胚胎成蠕虫状幼虫。继形态，胚胎孵化和幼虫L1在开始他们的直接环境中使用有益菌喂养。
因此胚胎伸长率是胚胎发育的后期阶段。它由沿其纵向轴线的胚胎的延伸和减少其横向直径的。这涉及皮下细胞形状的急剧改变。伸长分为早期和晚期。早期阶段开始于逗号阶段结束时，体壁肌肉开始在以W 1.75倍承包期ILD型（wt）的胚胎-对应于1.75倍的长度相比，非细长的胚胎的胚胎。在该阶段发生的形态发生的过程，主要由设在皮下细胞的顶磁极丝状肌动蛋白束（FB）的驱动其伸长沿胚胎2的前-后轴线的收缩驱动。 FB的收缩是通过控制肌球蛋白轻链的磷酸三激酶LET-502 / ROCK，MRCK-1和PAK-1 5。伸长的后期，开始时身体壁肌肉变得功能，并开始承包。它涉及从体内壁肌肉的背部和腹部皮下细胞机械传导信令和结束时动物孵化3。
伸长缺陷通常的特征在于由动物死于胚胎的百分率（胚胎致死性; EMB）和那些阻止其为L1幼虫（幼虫被捕表型的发展;吕a）和比重量显著缩短。发育停滞阶段的鉴定需要死胚显微镜观察并逮捕幼虫3-6。
据最近表明，一些基因，如Cdc42的/ Rac的调节和效应PIX-1和PAK-1，早期和晚期伸长3,7-期间控制形态发生的过程。我们最近还表明，形态发生过程早期伸长3 7期间沿胚的前-后轴线不同。这些发现促使新指标专门针对早期或晚期阶段的延伸和其他指标，可早在伸长沿前 – 后轴上胚胎的形态表征的发展。
这些新颖的方法包括在测量胚的长度在开始和早期伸长的末端以及它们的宽度，他广告和尾巴。7两个协议还制定了衡量初孵幼虫的长度，在L1阶段7同步。
胚胎的蛋壳保护它们免受碱性次氯酸盐处理而幼虫，成虫和存在于培养基的细菌通过处理溶解。然后，该处理用于从含有多数精心喂养成人8的非同步人口纯化胚胎。食物相克是用来初孵幼虫同步。测量这些幼虫的长度，然后用于检测伸长缺陷。这种测量优于在培养皿被捕幼虫的测量，因为幼虫从孵化非充分拉长胚胎可以恢复喂奶时“正常的长度”，但在缺乏食物时被捕将保持它们的尺寸减小。
在这里，我们提出详细的协议，使的乐测量延长使用时间推移DIC显微镜和图像分析（方案1）的胚胎，以及他们的头部的宽度和尾部NGTH。我们还提供了详细的协议，来测量使用图像分析（协议2）和流式细胞仪（协议3）同步幼虫的长度。
Protocol
Representative Results
Discussion
本协议描述的新的指标来表征早期胚胎和伸长率的后期阶段。
在部分1中，关键的步骤是细菌在垫中可能存在。将胚气密封闭垫和图像采集期间盖玻片之间。密封滑动时需要避免采集，持续两个多小时期间动物的干燥。据我们所知，没有用于安装滑动和盖玻片之间琼脂糖垫密封剂是透气的。因此，当大量的细菌（或胚胎）的存在于垫，它们可以在几个小时导致其过早死亡后缺?…
開示
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada and The Canada Foundation for Innovation. Thanks to Dr Paul Mains (University of Calgary, Calgary, Canada) for let-502(sb118ts) strain. Some of the strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).
Materials
| Agar | BioShop | AGR001.500 | |
| Agarose | Bioshop | AGA001.500 | |
| CaCl2 (calcium chloride) | Bio Basic Inc. | CT1330 | |
| Cholesterol | Sigma-aldrich | C8667 | |
| cleaning solution | union Biometrica | 300-5072-000 | |
| glass coverslips | Fisherbrand | 12-542B | |
| glass slides | Fisherbrand | 12-552-3 | |
| high fluorescent control particles | union Biometrica | 310-5071-001 | |
| K2HPO4 (potassium phosphate, dibasic) | Bio Basic Inc. | PB0447 | |
| KH2PO4 (potassium phosphate, monobasic) | Bio Basic Inc. | PB0445 | |
| MgSO4 (magnesium sulfate) | Sigma-aldrich | 230391 | |
| Na2HPO4( sodium phosphate, dibasic) | Bio Basic Inc. | SDB0487 | |
| NaCl (sodium chloride) | Bio Basic Inc. | DB0483 | |
| Pebeo Drawing Gum 45ml | pébéo | PDG033000 | any art/craft store |
| Peptone | BioShop | PEP403.500 | |
| Sheath buffer | union Biometrica | 300-5070-100 | |
| COPAS Biosort | union Biometrica |
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