在永久性幼虫特有的神经重构的C语言活体成像线虫
Summary
Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant ‘dauer’ juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.
Abstract
控制应激诱导的表型可塑性在动物的机制是频繁复杂和困难的体内研究。应力诱发塑性的一个典型的例子是C的持久期线虫 。 Dauers是低浓度的细菌性食物和高浓度永久性幼虫信息素的条件下形成的一种替代发育的幼虫期。 Dauers显示广泛的发育和行为的可塑性。例如，一套四内唇象限（IL2Q）神经元中持久形成进行了广泛可逆的重塑。利用众所周知的环境途径调节永久性幼虫条目，先前建立的方法，用于生产由大型液体线虫培养物粗永久性幼虫信息素被证实。用这种方法，对50000的浓度 – 液体培养物75000线虫/ ml的是足以产生一种高效的粗永久性幼虫信息素。粗素效价对射探测器由剂量 – 反应的生物测定rmined。最后，在持久形成用于体内的时间推移的IL2Qs的成像方法进行描述。
Introduction
表型可塑性，包括发育和行为的可塑性，可以适应恶劣的环境条件很重要，被认为是动力，发展1。℃。线虫是一种模式生物经常使用的开发和神经生物学研究。在理想情况下，C。在进入成年繁殖期通过四个幼虫阶段线虫的发展。然而，低粮食供应和人口密度高，长的条件下， 线虫可以接受的发育开关，进入一个长寿命和抗逆持久期2。 Dauers显示非dauers几个形态和行为上的差异而可能介导这一抗逆性。的环境线索和遗传途径调节永久性幼虫条目已经广泛地描述3，4，5，6，7，但控制个体的表型变化的分子机制发生ðuring持久形成是不太容易理解。
永久性幼虫特定表型的检测要求生产的永久性幼虫动物。这可以通过以下三种方式：1）从C的饥饿文化采摘线虫 ，2）使用的持久形成组成（DAF-C）突变体，3）诱导持久形成经过纯化信息素。这是比较简单的，从一个标准的NGM板有三挑dauers 线虫的人口已经耗尽了它的细菌的食物供应。在我们手中，一个标准的NGM板最初接种用50μlOP50 大肠杆菌 ，使其生长3天，随后接种3 – 4成人雌雄同体保 持在25℃，将用尽的一个星期内的食物供应和产生几千内的3天dauers（除了众多饥饿非dauers）。然而，这种方法是不令人满意的蜕皮到d中期间出现的审查过程奥尔。因此很难确定哪个几千动物在一个典型的饥饿板的正进入永久性幼虫。此外，使用饿死板提供了可能性进行同步。采用DAF-C突变允许同步和可靠的代dauers的。然而，没有保证特定的daf-C突变不会影响其他永久性幼虫特异性表型单独或与其他突变组合。
à永久性幼虫信息素的存在，最初是由Cassada和罗素暗示，后来被黄金和里德尔证实。该持久性信息素已被化学特点2，8，9，10，纯化信息素是由一个复杂的混合ascarosides，它以不同的形式规范双方的行为和发育过程9,11。使用粗永久性幼虫信息素的提取物可以用于可靠的控制异步同步dauers在野生型背景。 </P>
对神经系统和相关的神经胶质细胞在永久性幼虫幼体形成12，13，14进行改造，其中一些变化是不可逆的，如神经胶质细胞的永久性幼虫13，14在重塑，但其他的重塑事件后，恢复到良好的可逆性环境条件。枝晶树枝状，从单一的树突切换到multidendritic神经元和轴突重塑12：例如，一组4 IL2Q神经元中持久形成包括经过快速，可逆的神经重构。本文的方法允许可靠的体内成像应激诱导的神经可塑性的模式生物。提出了生产和粗永久性幼虫信息素的检测方法被先前描述和编译从几个来源4，8，15，16，17，18。
Protocol
Representative Results
Discussion
永久性幼虫特异性神经可塑性和其他永久性幼虫相关的形态学变化的检查需要控制和可靠的形成dauers要么通过基因突变（ 即，DAF-C突变体）或由野生型动物的接触信息素。而使用的基因突变的诱发dauers方便，它可能会混淆的结果。因此，与DAF-C突变收集的数据应该被随后确认与诱导暴露于永久性幼虫信息素进入持久期野生型动物。
的粗永久性幼虫信息素的生产相…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
I thank Dr. Maureen Barr, under whose supervision the dauer IL2 remodeling phenotype was initially characterized. Thanks to Dr. C. Britt Carlson for critical reading of the manuscript. Funding in the Barr lab was from the NIH (5R01DK59418) and postdoctoral fellowships from the USDA (2010-65106-20587) and the New Jersey Commission on Spinal Cord Research (CSCR12FEL004). Current funding for this work is from a University of Illinois Urbana-Champaign College of ACES FIRE grant.
Materials
| N2 C. elegans Bristol strain | Caenorhabditis Genetics Center | ||
| PT2660 myIs13[Pklp-6::gfp] III | Upon request from Schroeder lab | ||
| PT2762 myIs14[Pklp-6::gfp] V | Upon request from Schroeder lab | ||
| JK2868 qIs56[Plag-2::gfp] V | Caenorhabditis Genetics Center | ||
| OP50 E. coli | Caenorhabditis Genetics Center | ||
| Petri dishes | Tritech Research | 60 mm T3308, 35 mm T3501 | |
| NGM agar | Various | 3 g NaCl, 17 g agar, 2.5 g peptone in 975 mL H2O autoclaved cooled to 55°C followed by 1 mL of 5 mg/ml cholesterol in ethanol, 25 mL of 1 M KPO4 buffer (pH 6.0), 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4 | |
| S Media | Various | S media consists of S basal solution (1.46 g NaCl, 0.25g K2HPO4, 1.5 g KH2PO4, 0.25 ml of 5 mg/ml cholesterol in ethanol and H2O to 250 mL. Autoclaved.) To the S basal solution add 2.5 mL of potassium citrate pH 6.0 (2 g citric acid monohydrate, 29.4 g tri-potassium citrate monohydrate, H2O to 100 mL, autoclaved), 2.5 mL of Trace Metals Solution (1.86 g disodium EDTA, 0.69 g FeSO4 •7 H2O, 0.2 g MnCl2•4 H2O, 0.29 g ZnSO4 •7 H2O, 0.025 g CuSO4 •5 H2O, H2O to 1 liter. Autoclaved and stored in the dark), 0.75 mL of 1 M CaCl2, and 0.75 mL of 1 M MgSO4. | |
| Dauer pheromone plates for dose-response assay | Various | Mix 0.085 g Noble agar, 0.5 mL of 0.513 M NaCl and 5 µL of 5 mg/ml cholesterol dissolved in ethanol in each of 6x 15 mL culture tubes. Add sufficient crude pheromone to each tube to create a range of concentrations. Add H2O to bring the volume to 4.725 mL. Boil over a Bunsen burner until the agar is dissolved and then cool to 55 °C. Add 125 µL 1 M KPO4 buffer (pH 6.0), 50 µl 0.1 M CaCl2, 50 µL 0.1 M MgSO4 and 50 µl of 5 mg/ml streptomycin sulfate to each tube. Pour the contents of each tube into a 35 mm diameter Petri dish. Let the agar solidify at room temperature overnight (if longer, store in enclosed container to prevent excessive drying). | |
| Microsphere beads 0.1 micron | Polysciences Inc. | 00876-15 | |
| M9 buffer | Various | Mix 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 liter. Sterilize by autoclaving |
Referenzen
- West-Eberhard, M. J. . Developmental Plasticity and Evolution. , (2003).
- Cassada, R. C., Russell, R. L. The dauerlarva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans. Dev. Biol. 46, 326-342 (1975).
- Riddle, D. L., Albert, P. S., Riddle , D. L., Blumenthal, T., Meyer, B. J., Priess, J. R. . C. elegans II. , 739-768 (1998).
- Golden, J. W., Riddle, D. L. The Caenorhabditis elegans dauer larva: developmental effects of pheromone, food, and temperature. Dev. Biol. 102, 368-378 (1984).
- Riddle, D. L., Wood, W. B. . The Nematode C. elegans. , 393-412 (1988).
- Fielenbach, N., Antebi, A. C. elegans dauer formation and the molecular basis of plasticity. Genes Dev. 22, 2149-2165 (2008).
- Hu, P. J. . WormBook. , 1-19 (2007).
- Golden, J. W., Riddle, D. L. A pheromone influences larval development in the nematode Caenorhabditis elegans. Science. 218, 578-580 (1982).
- Butcher, R. A., Fujita, M., Schroeder, F. C., Clardy, J. Small-molecule pheromones that control dauer development in Caenorhabditis elegans. Nature Chemical Biology. 3, 420-422 (2007).
- Jeong, P., et al. Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone. Nature. 433, 541-545 (2005).
- Srinivasan, J., et al. A blend of small molecules regulates both mating and development in Caenorhabditis elegans. Nature. 454, 1115-1118 (2008).
- Schroeder, N., et al. Dauer-Specific dendrite arborization in C. regulated by KPC-1/Furin. Curr. Biol. 16, 1527-1535 (2013).
- Procko, C., Lu, Y., Shaham, S. Glia delimit shape changes of sensory neuron receptive endings in C. elegans. Development. 138, 1371-1381 (2011).
- Albert, P. S., Riddle, D. L. Developmental alterations in sensory neuroanatomy of the Caenorhabditis elegans dauer larva. J. Comp. Neurol. 219, 461-481 (1983).
- Vowels, J. J., Thomas, J. H. Genetic analysis of chemosensory control of dauer formation in Caenorhabditis elegans. Genetik. 130, 105-123 (1992).
- Vowels, J. J., Thomas, J. H. Multiple chemosensory defects in daf-11 and daf-21 mutants of Caenorhabditis elegans. Genetik. 138, 303-316 (1994).
- Ailion, M., Thomas, J. H. Dauer formation induced by high temperatures in Caenorhabditis elegans. Genetik. 156, 1047-1067 (2000).
- Neal, S. J., Kim, K., Sengupta, P. . Pheromone Signaling. , 273-283 (2013).
- Brenner, S. The genetics of Caenorhabditis elegans. Genetik. 77, 71-94 (1974).
- Ouellet, J., Li, S., Roy, R. Notch signalling is required for both dauer maintenance and recovery in C. elegans. Development. 135, 2583-2592 (2008).
- Blelloch, R., et al. The gon-1 gene is required for gonadal morphogenesis in Caenorhabditis elegans. Dev. Biol. 216, 382-393 (1999).
- Fang-Yen, C., Gabel, C. V., Samuel, A. D., Bargmann, C. I., Avery, L. Laser microsurgery in Caenorhabditis elegans. Methods Cell Biol. 107, (2012).
- Nelson, F. K., Riddle, D. L. Functional study of the Caenorhabditis elegans secretory-excretory system using laser microsurgery. J. Exp. Zool. 231, 45-56 (1984).
- Sulston, J. E., Horvitz, H. R. Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev. Biol. 56, 110-156 (1977).
- Singh, R., Sulston, J. Some observations on moulting in Caenorhabditis elegans. Nematologica. 24, 63-71 (1978).
