形态分析果蝇幼虫外周感觉神经元树突和轴突使用遗传马赛克
Summary
感觉神经元的树突分枝<em>果蝇</em>幼虫周围神经系统是有用的模型，以澄清一般和神经元类特定的神经细胞分化的机制。我们提出了一个实用指南生成和分析树突状树枝状的神经元遗传马赛克。
Abstract
神经系统的发育需要的神经元的地位和身份，准确的神经元类的特定的树突状发展和轴突布线，正确规范。最近的树突状树枝状（DA） 的果蝇幼虫周围神经系统（PNS）的感觉神经元已成为强大的遗传模型，在其中阐明神经元分化的一般和特定类的机制。有四个主要的DA神经元类（I – IV）1 。他们被命名为了增加枝蔓乔木的复杂性，并在其分化2-10的基因控制类特定的差异。 DA感官系统是一个实际的模型研究的树突状形态 11-13的控制背后的分子机制，因为：1）它可以利用强大的基因在果蝇中的可用工具，2）的优势，DA神经元枝蔓乔木展开在下方的光骑士尺寸只有2AR幼虫角质层很容易地可视化体内的高分辨率，3）类特定的树突形态的多样性有利于比较分析，发现控制简单与高度分支的树突树形成的关键要素，以及4）树突状乔木的定型不同的DA能神经元的形状有利于形态的统计分析。
DA神经元活动的修改输出的一个幼虫的运动中枢模式发生器 14-16 。不同的DA神经元类有不同的感觉方式，和它们的激活会引起不同的行为反应14,16-20。此外，不同类别发送到果蝇幼虫中枢神经系统的腹 神经索（VNC） 的 21轴索预测刻板。这些预测终止两个DA神经元感觉方式和位置在体壁的树突状领域7,22地形意见书23。因此，DA的轴突预测的检查可用于澄清的内在机制， 地形测绘7,22,23，以及一个简单的电路调节幼虫运动14-17的接线。
我们在座的生成和分析遗传马赛克24标记的DA通过MARCM（马赛克Repressible细胞标记分析）1,10,25和FLP – 22,26,27技术（在图1总结）神经元的实用指南。
Protocol
1.Preparation试剂准备钙离子免费HL3.1生理盐水28。 （毫米）：70氯化钠，氯化钾，氯化镁2 20 10 3，碳酸氢钠5 HEPES，115蔗糖，和5海藻糖; pH值7.2。过滤消毒，并储存于4 ° C。 注意：CA + +免费的解决方案可以防止肌肉收缩，在清扫。 设为聚- L -赖氨酸（PLL）盖玻片。 4.2毫升水溶解100毫克PLL和300μL分装在Eppendorf管中，并冻?…
Discussion
果蝇幼虫DA神经元模型提供了一个优良的遗传系统调查机制，控制神经元的形态和电路形成。 MARCM是一般用于标签和产生突变的DA神经元克隆。 MARCM，我们使用一个泛神经（ 如Gal4 的 C155）或DA神经元特异性司机。使用泛神经的驱动程序，它可以直接使用公共储备中心广泛使用的几个股票。然而，使用DA神经元的特定的驱动程序可以是有利的，因为标记克隆不会产生在中?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢理化学研究所的资金。我们也感谢Cagri Yalgin，加Delandre，和周杰伦帕里什讨论遗传和免疫组化协议。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| SZX16 fluorescence dissection microscope (with GFPHQ filter) | Olympus | SZX16 | |
| Live Insect Forceps | FST | 26030-10 | |
| 26mm x 76mm depression slide glass | Toshinriko Co. | T8-R004 | |
| Sylgard 184 (or Silpot 184) | Dow Corning | 3097358-1004 | |
| Poly-L-lysine | Sigma | P-1524 | This product has proven most effective |
| DPX mounting medium | Sigma | 44581 | |
| Rabbit anti-GFP | Invitrogen | A-11122 | Dilution 1:500 |
| Rat anti-CD8 | Caltag | 5H10 | Dilution 1:200 |
| Mouse anti-CD2 | AbD serotec | MCA443R | Dilution 1:700 |
| Mouse anti-Fasciclin2 | DSHB | 1D4 | Dilution 1:10 |
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Tags
Morphological AnalysisDrosophila LarvalPeripheral Sensory NeuronDendritesAxonsGenetic MosaicsNervous System DevelopmentNeuron PositionNeuron IdentityDendritic DevelopmentAxonal WiringDendritic Arborization (DA)Sensory NeuronsPeripheral Nervous System (PNS)Genetic ModelsNeuron DifferentiationClass-specific MechanismsDendritic MorphologyMolecular MechanismsFruit FlyLarval CuticleHigh Resolution VisualizationComparative AnalysisMorphometric Statistical AnalysisDA Neuron ActivityLocomotion Central Pattern Gene
Citazione di questo articolo
Karim, M. R., Moore, A. W. Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics. J. Vis. Exp. (57), e3111, doi:10.3791/3111 (2011).