Fluorescent Beads Are a Versatile Tool for Staging Caenorhabditis elegans in Different Life Histories.
Fluorescent Beads Are a Versatile Tool for Staging Caenorhabditis elegans in Different Life Histories.
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DOI：
10.1534/g3.116.030163
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发表时间：
2016-07-07
期刊：
影响因子：
--
通讯作者：
Karp X
中科院分区：
文献类型：
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作者：
Nika L;Gibson T;Konkus R;Karp X
秀丽隐杆线虫的精确分期对不同环境条件下的发育研究至关重要。在有利条件下，幼虫连续发育，以蜕皮期为间隔，分为四个幼虫期。利用转基因技术和蜕皮报告者可以区分蜕皮和间皮幼虫，因此需要菌株构建，或仔细观察个体的咽泵或行为静止。在不利条件下，幼虫可进入抗胁迫和发育受阻的大幼虫期。鉴定道尔幼虫是基于它们耐受洗涤剂选择的能力，排除了鉴定恢复中的动物或道尔形态发生缺陷的突变体。在这里，我们描述了一种简单的方法来区分蜕皮幼虫或蜕皮幼虫与中间幼虫，绕过了现有方法的局限性。荧光乳胶珠与细菌食物源混合，由换壳幼虫和成虫摄入。蜕皮幼虫和蜕皮幼虫不进食，因此消化道中缺少珠子。珠子的存在可以使用放大率低至100倍的解剖显微镜来确定，或者通过使用蠕虫分选器进行高通量实验。我们发现，持续发育的无珠幼虫表现出蜕皮的特征，包括mlt-10::gfp蜕皮标记的表达和咽泵的缺乏。此外，通过三种常用方法中的任何一种产生的野生型和突变型道尔幼虫都可以通过缺乏珠子来准确识别。重要的是，该方法在sds敏感的突变体背景下是有效的，并且可以识别恢复期的dawer幼虫，这是一个没有其他阳性选择方法的阶段。
Precise staging of Caenorhabditis elegans is essential for developmental studies in different environmental conditions. In favorable conditions, larvae develop continuously through four larval stages separated by molting periods. Distinguishing molting from intermolt larvae has been achieved using transgenes with molting reporters, therefore requiring strain constructions, or careful observation of individuals for pharyngeal pumping or behavioral quiescence. In unfavorable conditions, larvae can enter the stress-resistant and developmentally arrested dauer larva stage. Identifying dauer larvae has been based on their ability to withstand detergent selection, precluding identification of recovering animals or of mutants with defects in dauer morphogenesis. Here, we describe a simple method to distinguish molting larvae or dauer larvae from intermolt larvae that bypasses the limitations of current methods. Fluorescent latex beads are mixed with the bacterial food source and ingested by intermolt larvae and adults. Molting and dauer larvae do not feed, and therefore lack beads in their digestive tract. The presence of beads can be determined using a dissecting microscope at magnifications as low as 100 ×, or by using a wormsorter for high-throughput experiments. We find that continuously developing bead-lacking larvae display hallmarks of molting, including expression of the mlt-10::gfp molting marker and a lack of pharyngeal pumping. Furthermore, wild-type and mutant dauer larvae produced by any of three common methods are accurately identified by a lack of beads. Importantly, this method is effective in SDS-sensitive mutant backgrounds and can identify recovering dauer larvae, a stage for which there is no other method of positive selection.