为什么量化事项：表型的表征在<em>果蝇</em>幼虫神经肌肉接头
Summary
Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.
Abstract
形态上的大多数研究依赖于如何解剖性状是由特定的基因和遗传途径的破坏影响的定性描述。定量描述很少进行的，虽然基因操作产生一系列表型的影响和变化即使在对照组中的个体观察。越来越多的证据表明，形态，大小和细胞器的位置，起到细胞功能和生存以前怀才不遇，但根本性的作用。在这里，我们在果蝇幼虫神经肌肉接头（NMJ）进行表型的定量分析提供一步一步的指示。我们使用多种可靠的免疫组化标记与生物成像技术和形态分析，研究特定的细胞过程的基因突变的影响相结合。特别是，我们专注于表型的定量分析影响的形态，大小，n的位置uclei内果蝇幼虫的横纹肌。 果蝇幼虫NMJ是一个有价值的实验模型来研究的结构和神经肌肉系统的功能，无论是在健康和疾病的分子机制。然而，我们在这里描述的方法可扩展到其他系统也是如此。
Introduction
Qualitative analysis restricts the focus of most experimental studies to the examination of genetic manipulations leading to large phenotypic effects or to phenotypes that are not amenable to quantification, e.g., absence/presence. Quantification of phenotypes is not usually performed and variations present within members of a phenotypic group are not taken into consideration. Additionally, without mathematical descriptions of morphologies, it may be difficult to determine whether fine scale phenotypic changes are the result of genetically induced alterations or whether observed changes are simply due to random fluctuations.
We propose that for an accurate and unbiased analysis of phenotypic defects due to gene disruption, quantitative methodologies should accompany more traditional qualitative approaches. Quantitative evaluation of phenotypes is particularly beneficial for structures such as synapses that present a high degree of variability due to their intrinsic morphological and functional plasticity. We have selected examples of quantitative analyses applied to areas of investigation with which we are most familiar, namely the Drosophila larval neuromuscular junctions (NMJs). However, concepts and principles apply equally well to other experimental systems.
The larval NMJ is an excellent model system to study synaptic development and function because of the highly stereotyped nature of its structure. Each hemi-segment of the larval neuromuscular system contains 32 identifiable motor neurons forming synaptic contacts with their postsynaptic target muscle. Every hemi-segment also contains a fixed number of muscles visible as multinucleated fibers attached to the internal surface of the cuticle1. Another advantage of using the larval neuromuscular system is the power and versatility of Drosophila genetics that easily allows the generation of a number of mutant alleles and the possibility to modify gene expression in a time- and tissue-restricted manner. Finally, 75% of the human genes causing a disease have an evolutionarily conserved orthologue in Drosophila2. Indeed, entire genetic pathways are conserved between flies and humans. Because of this, the Drosophila larval neuromuscular system is a very popular experimental model to elucidate the molecular mechanisms underlying a number of human diseases including amyotrophic lateral sclerosis (ALS)1.
Here we show that the availability of several reliable immuno-histochemical markers, combined with bio-imaging techniques and accurate morphometric analyses can describe anatomical traits that are likely to play an important functional role3,4,5. Among the cellular processes that are amenable to quantitative analyses, we focus on changes in shape, size and position of intracellular structures such as the nuclei. All these are processes that we know very little about.
The challenge for molecular geneticists in the coming decades will be to extend our current knowledge by analyzing the effect of genetic mutations that produce very subtle phenotypic defects. Quantitative methodologies that allow researchers to meticulously explore the effects of genetic mutations can provide a more comprehensive understanding of how genotypes relate to phenotypes, especially for poorly understood cellular processes.
Protocol
Representative Results
Discussion
在过去，中和实验组之间的形态差异很少考虑。然而，定量方法的应用现在已经成为在形态和解剖形式的数学描述的比较研究规范进行计算。采用定量的评估特定的细胞过程的遗传操作的影响进行分析，拿在提高我们检测形态变化能力，改善与这些变化中描述的准确性的承诺。此外，量化数据的统计分析使我们能够评估表型之间观察到的差异是否显著。
在横纹肌，细胞核表现出明显的圆形结构，并沿肌纤维均匀地分布。尽管建立和保持的大小，形状和原子核的结构的分子机制尚不清楚，这些核特征可能吨Ø在控制肌肉功能的基础性作用。事实上，几个肌病是由突变基因的肌肉内调节原子核的形态和位置造成的。形状和小区内的核的分配的功能的重要性并不限于肌肉。越来越多的证据表明，核的缺陷也与神经变性疾病如帕金森氏病18,24相关联。此外，我们开始明白，形态，大小和其他细胞器的细胞内分布，包括内质网和线粒体可能有功能的后果。例如，在线粒体形态的改变与神经性疾病如视神经萎缩型-1（OPA1）和腓骨肌萎缩2A型神经病25相关联。
协助阐明潜在这些重要过程的分子机制的过程中，我们建议高分辨率共焦结合与成像软件和形态数据分析，以定量地评价遗传操作如何肌纤维内影响的形状，大小，与原子核的位置。电源和果蝇遗传学的多功能性加上在果蝇幼虫的神经肌肉系统的高度定型性质使幼虫NMJ的实验模型特别适合这种类型的分析。在幼虫NMJs，表型分析可以在单个突触分辨率允许其中多个NMJs可以在同一飞行中进行研究，甚至同一识别NMJ可以不同基因型3,4的苍蝇之间进行比较精确的形态测定分析来进行。
在果蝇幼虫NMJ核位置，形状和大小的表型特征通过与突出的肌肉内的肌肉和细胞核抗体进行解剖NMJs的免疫染色开始。在协议THI概述造纸，肌细胞核用针对核纤层蛋白，核膜的标志多克隆抗体染色，用核标记突出特定DVAP弄脏整个肌肉核内部和抗体。在这些实验中使用的核纤层蛋白抗体麻烦由Paul费舍尔19-22提供，但可以使用的抗拉明抗体的替代来源。另外，许多特异于核膜其他抗体是市售的。最后，核标记物，如DAPI和碘化丙锭，也可同时肌肉可以通过用抗肌动蛋白或抗微管蛋白抗体染色可视化。如果比那些在本实验过程中使用的其他抗体使用的，免疫染色协议将需要额外的步骤，其中的固定条件和工作浓度为新的抗体将需要优化。在这个协议中，特别是当体积渲染需要分析一个关键的步骤，是第Ë安装在幻灯片上的样品。在这种情况下，以包括载玻片和盖玻片使得试样没有得到挤压之间的间隔是重要的。绕在盖玻片的两侧滑动缠绕纤维素带的三个带代表制作隔板的简单方法。
虽然被用于2D图像ImageJ的，大部分的3D多通道图像在本文分析介绍，通过使用，因为其内部的可用性了Imaris完成。然而，任何其他类似的商业软件包可用于这些应用。
有几个开源（例如，ImageJ的，CellProfiler，Vaa3D，冰爽，KNIME等）和商业软件平台，可用于共聚焦图像的分析。 ImageJ的26日 ，由美国国立卫生研究院或其更多加强版，被称为斐济27免费软件，具有可用于全球成像一个省交通大量进口过滤器，宏和插件TY。大多数这些插件都集中在加工的片逐片方式的信息。也有可用于多通道的3D图像的可视化和分析的插件。然而，他们通常被设计为一个特定的任务，用户可根据需要这些插件扩展或适应自己的需要。在另一方面，商业平台的目标相对缺乏经验的用户，而且往往集中在易于使用，具有令人难以置信的高速图像处理任务覆盖面广。
在这个协议中所概述的定量表型分析的实验程序一起，可以协助在阐明的分子机制控制细胞器的形态和细胞内的分布。但是，这种方法具有一个特定终点分析这些过程的明显的限制。控制的形态和细胞器的分配过程中可能是非常动态的，并不仅不同细胞TY之间变化PES而且取决于发育或生理状态在同一小区内。这一分析的进一步实施将通过时间推移成像，允许在细胞器的形态和位置的变化随着时间的推移进行监控来表示。
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
We are grateful to Dr. Andrea Chai for her insightful comments on the manuscript. This work was supported by the Wellcome Trust (grant number: Pennetta8920) and by the Motor Neuron Disease Association (grant number: Pennetta6231).
Materials
| Micro-Forceps 0.3×0.25 mm | Fine Science Tools | 11030-12 | |
| Sylgard dissection plates | SIGMA-ALDRICH | 76103 | Mix the pre-weighed elastomer base with curing agent. Poor the mixture into a 5 cm Petri dish. Let cure it at 60ºC for at least 24 hours |
| Stainless/Steel Minutien Pins 0.1 mm diameter | Fine Science Tools | 26002-10 | |
| Microdissection scissors (ultra-fine) | Fine Science Tools | 15200-00 | |
| 1x PBS (Phosphate Buffered Saline). Composition: 3mM NaH2PO4, 7mM Na2HPO4, 130mM NaCl, pH 7 | NaH2PO4 (SIGMA-S8282), Na2HPO4 (SIGMA-S7907), NaCl (SIGMA-S7653) | ||
| Bouin's Solution. Composition: Picric Acid, Formaldehyde, Acetic Acid (15:10:1) | Picric Acid (SIGMA 197378), Formaldehyde (F8775), Acetic Acid (SIGMA-1005706) | ||
| 1x PBT (Phosphate Buffered Saline with Triton). 1xPBS +0.1% Triton | Triton-X100. SIGMA-T8787 | ||
| Normal Goat Serum | SIGMA | G9023 | |
| Guinea Pig anti-DVAP antibody | Provided by Dr. Giuseppa Pennetta (University of Edinburgh, UK). Use at !:200 dilution in 5% NGS | ||
| Rabbit anti-HRP (Horseradish peroxidase) | Jackson ImmunoResearch | 123-065-021 | Use at 1:500 dilution in PBT containing 5% NGS |
| Rabbit anti-Lamin antibody | Provided by Dr. Paul Fisher (State Univeristy of New York at Stony Brook). Use at 1:500 dilution in PBT containing 5% NGS | ||
| TO-PRO-3 | Molecular Probes | T3605 | |
| Goat anti-rabbit antibody, Alexa Fluor488, conjugated | Jackson ImmunoResearch | bs-295G-A555-BSS | Use at 1:500 dilution in PBT containing 5% NGS |
| Goat anti-guinea pig IgG antibody, Cy3, conjugated | Jackson ImmunoResearch | bs-0358G-Cy3-BSS | Use at 1:500 dilution in PBT containing 5% NGS |
| Vectashield mounting medium for fluorescence | Vector laboratories | H-1000 |
Referenzen
- Collins, C. A., DiAntonio, A. Synaptic development: insights from Drosophila. Curr. Opin. Neurobiol. 17 (1), 35-42 (2007).
- Fortini, M. E., Skupski, M. P., Boguski, M. S., Hariharan, I. K. A survey of human disease gene counterparts in the Drosophila genome. J. Cell Biol. 150 (2), 23-30 (2000).
- Ramachandran, P., Budnik, V. Dissection of Drosophila larval body-wall muscles. Cold Spring Harb Protoc. (8), (2010).
- Brent, J. R., Werner, K. M., McCabe, B. D. Drosophila larval NMJ dissection. JoVE. (24), (2009).
- Brent, J., Werner, K., McCabe, B. D. Drosophila larval NMJ immunohistochemistry. JoVE. (25), (2009).
- Ramachandran, P., Budnik, V. Immunocytochemical staining of Drosophila larval body-wall muscles. Cold Spring Harb Protoc. (8), (2010).
- Ling, S. C., Polymenidou, M., Cleveland, D. W. Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron. 79 (3), 416-438 (2013).
- Nishimura, A. L., et al. A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am J Hum Genet. 75 (5), 822-831 (2004).
- Chen, H. J., et al. Characterization of the properties of a novel mutation in VAPB in familial amyotrophic lateral sclerosis. J Biol Chem. 285 (51), 40266-40281 (2010).
- Funke, A. D., et al. The P56S mutation in the VAPB gene is not due to a single founder: the first European case. Clin Genet. 77 (3), 302-303 (2010).
- Millecamps, S., et al. SOD1,ANG,VAPB,TARDBP, and FUS mutations in familial amyotrophic lateral sclerosis: genotype-phenotype correlations. J Med. Genet. 47 (8), 554-560 (2010).
- Landers, J. E., et al. New VAPB deletion variant and exclusion of VAPB mutations in familial ALS. Neurology. 70 (14), 1179-1185 (2008).
- Van Blitterswijk, M., et al. VAPB and C9orf72 mutations in 1 familial amyotrophic lateral sclerosis patient. Neurobiol Aging. 33 (12), 2951-2954 (2012).
- Brand, A. H., Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 118 (2), 401-415 (1993).
- Budnik, V., et al. Regulation of synapse structure and function by the Drosophila tumor suppressor gene dlg. Neuron. 17 (4), 627-640 (1996).
- Sanhueza, M., Zechini, L., Gillespie, T., Pennetta, G. Gain-of-function mutations in the ALS8 causative gene VAPB have detrimental effects on neurons and muscles. Biol Open. 3 (1), 59-71 (2014).
- Worman, H. J., Ostlund, C., Wang, Y. Diseases of the nuclear envelope. Cold Spring Harb Perspect Biol. 2 (2), a000760 (2010).
- Liu, G. H., et al. Progressive degeneration of human neural stem cells caused by pathogenic LRRK2. Nature. 491 (7425), 603-607 (2012).
- Pennetta, G., Hiesinger, P. R., Fabian-Fine, R., Meinertzhagen, I. A., Bellen, H. J. Drosophila VAP-33A directs bouton formation at neuromuscular junctions in a dosage-dependent manner. Neuron. 35 (2), 291-306 (2002).
- Fisher, P. A., Berrios, M., Blobel, G. Isolation and characterization of a proteinaceous subnuclear fraction composed of nuclear matrix, peripheral lamina, and nuclear pore complexes from embryos of Drosophila melanogaster. J Cell Biol. 92 (3), 674-686 (1982).
- Smith, D. E., Fisher, P. A. Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J Cell Biol. 99 (1), 20-28 (1984).
- Smith, D. E., Gruenbaum, Y., Berrios, M., Fisher, P. A. Biosynthesis and interconversion of Drosophila nuclear lamin isoforms during normal growth and in response to heat shock. J Cell Biol. 105 (2), 771-790 (1987).
- Puckelwartz, M. J., et al. Disruption of nesprin-1 produces an Emery Dreifuss muscular dystrophy-like phenotype in mice. Hum Mol Genet. 18 (4), 607-620 (2009).
- Tsujikawa, M., Omori, Y., Biyanwila, J., Malicki, J. Mechanism of positioning the cell nucleus in vertebrate photoreceptors. Proc Natl Acad Sci USA. 104 (37), 14819-14824 (2007).
- Westermann, B. Mitochondrial fusion and fission in cell life and death. Nat Rev Mol Cell Biol. 11 (12), 872-884 (2010).
- Schneider, C. A., Rasband, W. S., Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 9 (7), 671-675 (2012).
- Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 9 (7), 676-682 (2012).
