在脑切片检测轴突本地化mRNAs的高空间分辨率<em>原位</em>杂交
Summary
RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.
Abstract
的mRNA经常局限于脊椎动物轴突和当地翻译所需的轴突寻路或在开发和维护，修理或神经变性postdevelopmental周期分支。高通量分析最近透露，轴突有一个更加动态和复杂的转录于此前预期。这些分析，但大多已在培养的神经元轴突的地方可以从躯体树突状车厢被孤立进行。这几乎是不可能实现这种隔离在体内整个组织。因此，为了验证的mRNA和它们的功能关联的募集在整体动物，转录组分析，最好应与技术，允许mRNA的原位可视化相结合。最近，已经开发出新颖的ISH技术，检测的RNA在单分子水平。当分析mRNA的亚细胞定位，这是特别重要由于本地化的RNA通常出现在较低的水平。在这里，我们描述的轴突本地化的mRNA使用一种新的超灵敏RNA原位杂交技术检测两个协议。使用荧光免疫组织化学和组织学染料，以验证ATF4 mRNA的招募到轴突在体内成熟小鼠和人的大脑我们结合RNAscope ISH与轴突染液。
Introduction
轴突mRNA的招聘和本地翻译的轴突能够在一个时间和空间的方式急响应1到胞外刺激。轴突内蛋白合成被最好地理解在神经发育的上下文中，其中它在生长锥行为2-8关键作用，轴突寻路9-11和逆行信令12,13。但到目前为止很少有关于轴突蛋白合成后发育神经元的功能意义时，轴突mRNA和核糖体水平都大大降低14,15。成熟脊椎动物轴突长期以来被认为是不活动的平移16。然而，最近的研究表明，本地翻译病理状态下重新激活在成熟轴突。例如，mRNA的一个子集被招募到再生轴突以下神经损伤和轴突内蛋白质合成所需的这些轴突17的正确再生。此外，集团公顷■证明特异mRNAs被招募到轴突后局部暴露于阿耳茨海默氏病的肽Aβ1-42，和转录因子ATF4的本地翻译需要从轴突传播的Aβ1-42的神经变性影响神经元胞体18。最后，高通量分析已经表明成熟轴突具有更复杂和动态的转录比预期18-21，特别是在病理状况。根据这些研究，一个高度敏感和特异的方法来检测在成年神经系统轴突本地化的mRNA是必要的。
许多关于基因招募和成熟轴突当地的翻译工作已经对培养的神经元被执行。这是特别真实对于转录分析自专门培养方法存在允许从躯体树突隔室18-20轴突的隔离。虽然这些研究都给予宝贵的插件飞行进入成熟轴突当地翻译的作用，问题是否培养的神经元忠实代表情况在体内或mRNA的招聘是轴突以培养条件的一种适应性反应仍然是开放的。很少有研究提供了表达的招聘证据成熟轴突在体内 。例如，所述转录物编码的嗅蛋白在成年感觉神经元22轴突已经检测。含有的β肌动蛋白的mRNA的3'非编码区的转基因被输送到在周围和中枢神经系统的神经元的轴突在小鼠和发育阶段23之后被局部翻译。拉明B2 mRNA定位到视网膜轴突Xenopues蟾蝌蚪和枯竭影响轴突发育后21轴突维修。干扰与细胞色素C氧化IV mRNA的编码的轴突运输改变鼠标的行为24。最后，ATF4的mRNA在成年找到的小鼠和人类大脑中的Aβ1-42的范围内xons诱导神经退行性变18。
高通量转录分析已被证明是有用的，以确定在分离的轴突在体外的mRNA型材但对体内研究的限制，因为在整个组织轴突从未发现在隔离但间杂有神经元细胞体，神经胶质细胞和其它细胞类型。因此，这样的分析必须与确认的mRNA的亚细胞定位的成像技术相结合。 原位杂交（RNA ISH）的RNA可在细胞和组织中的特定RNA序列的检测和可视化。然而，原始的RNA原位杂交测定法是只适合识别的高丰度的RNA 25，这是很少对轴突本地化的mRNA的情况。在过去的十年里越来越多的努力已投入开发新技术，使mRNA的检测在单分子水平<suP> 25,26。例如，Singer和同事开发ISH探针来检测的mRNA在单细胞，包括在5个非重叠的荧光标记的50聚体（详情参见27）。上述技术和此处描述的之间的主要区别是，以后使用20双Z型结构（未线性）探针一般是针对〜1kb的兴趣确保特异性和低背景水平的RNA的区域。探针杂交，然后与最终荧光标记或缀合的酶，使显色反应，前置放大器和功率放大器序列。这些扩增步骤改善信噪比相比其他ISH技术28。在这里，我们将介绍使用RNAscope结合无论是与免疫细胞化学荧光或组织学染料使轴突复染两种协议。这两个协议都适合可视化ATF4 mRNA在成年莫轴突本地化使用和人类大脑。
Protocol
Representative Results
Discussion
在这份报告中，我们描述了轴突本地化ATF4 mRNA的检测采用了高分辨率的ISH技术。这些和以前发表的研究报告表明，该技术是与基于抗体的蛋白质检测的组织中或甚至整个胚胎33兼容。重要的是，它最近已被使用于海马神经元34的树突内检测电弧的mRNA。它也可以与组织学染料对组织染色组合。最后，这是适合于同时检测多个靶RNA 28,30,33。这些结果例证的高分辨?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Alzheimer’s Association (NIRG-10-171721; to U.H.), National Institute of Mental Health (MH096702; to U.H.), National Institute of Neurological Disorders and Stroke (NS081333; to C.M.T.), and pilot study awards from the National Institute on Aging-funded Alzheimer’s Disease Research Center at Columbia University (AG008702; to J.B. and Y.Y.J.) that also supports the New York Brain Bank. We thank members of the Hengst laboratory for comments and discussions
Materials
| custom probe targeting residues 20-1381 of the mouse Atf4 mRNA (NM_009716) | Advanced Cell Diagnostics | – | probe |
| custom probe targeting residues 15-1256 of the human ATF4 mRNA (NM_001675.2) | Advanced Cell Diagnostics | – | probe |
| negative control probe-DapB | Advanced Cell Diagnostics | 310043 | probe |
| positive control probe-mouse Polr2A (optional) | Advanced Cell Diagnostics | 312471 | probe |
| positive control probe-human PPIB (optional) | Advanced Cell Diagnostics | 313901 | probe |
| RNAscope Fluorescent Multiplex Reagent Kit (for fluorescence detection) | Advanced Cell Diagnostics | 320850 | In situ hybridization kit |
| RNAscope 2.0 HD Reagent Kit – BROWN (for chromogenic detection) | Advanced Cell Diagnostics | 310035 | In situ hybridization kit |
| Goat polyclonal anti-ChAT antibody | Millipore | AB144P | |
| Luxol Fast Blue-Cresyl Echt Violet Stain Kit | American MasterTech | KTLFB | |
| Clearify clearing agent (xylene substitute) | American MasterTech | CACLEGAL | |
| ProLong Gold mounting medium with DAPI | Life Technologies | P36935 | |
| DPX mounting medium | Sigma | 6522 |
Riferimenti
- Jung, H., Yoon, B. C., Holt, C. E. Axonal mRNA localization and local protein synthesis in nervous system assembly, maintenance and repair. Nat. Rev. Neurosci. 13, 308-324 (2012).
- Campbell, D. S., Holt, C. E. Chemotropic responses of retinal growth cones mediated by rapid local protein synthesis and degradation. Neuron. 32, 1013-1026 (2001).
- Wu, K. Y., et al. Local translation of RhoA regulates growth cone collapse. Nature. 436, 1020-1024 (2005).
- Piper, M., et al. Signaling mechanisms underlying Slit2-induced collapse of Xenopus retinal growth cones. Neuron. 49, 215-228 (2006).
- Yao, J., Sasaki, Y., Wen, Z., Bassell, G. J., Zheng, J. Q. An essential role for β-actin mRNA localization and translation in Ca2+-dependent growth cone guidance. Nat. Neurosci. 9, 1265-1273 (2006).
- Hengst, U., Deglincerti, A., Kim, H. J., Jeon, N. L., Jaffrey, S. R. Axonal elongation triggered by stimulus-induced local translation of a polarity complex protein. Nat. Cell Biol. 11, 1024-1030 (2009).
- Gracias, N. G., Shirkey-Son, N. J., Hengst, U. Local translation of TC10 is required for membrane expansion during axon outgrowth. Nat. Commun. 5, 3506 (2014).
- Preitner, N., et al. APC Is an RNA-Binding Protein, and Its Interactome Provides a Link to Neural Development and Microtubule Assembly. Cell. 158, 368-382 (2014).
- Brittis, P. A., Lu, Q., Flanagan, J. G. Axonal protein synthesis provides a mechanism for localized regulation at an intermediate target. Cell. 110, 223-235 (2002).
- Tcherkezian, J., Brittis, P. A., Thomas, F., Roux, P. P., Flanagan, J. G. Transmembrane receptor DCC associates with protein synthesis machinery and regulates translation. Cell. 141, 632-644 (2010).
- Colak, D., Ji, S. J., Porse, B. T., Jaffrey, S. R. Regulation of axon guidance by compartmentalized nonsense-mediated mRNA decay. Cell. 153, 1252-1265 (2013).
- Cox, L. J., Hengst, U., Gurskaya, N. G., Lukyanov, K. A., Jaffrey, S. R. Intra-axonal translation and retrograde trafficking of CREB promotes neuronal survival. Nat. Cell Biol. 10, 149-159 (2008).
- Ji, S. J., Jaffrey, S. R. Intra-axonal translation of SMAD1/5/8 mediates retrograde regulation of trigeminal ganglia subtype specification. Neuron. 74, 95-107 (2012).
- Bassell, G. J., Singer, R. H., Kosik, K. S. Association of poly(A) mRNA with microtubules in cultured neurons. Neuron. 12, 571-582 (1994).
- Kleiman, R., Banker, G., Steward, O. Development of subcellular mRNA compartmentation in hippocampal neurons in culture. J. Neurosci. 14, 1130-1140 (1994).
- Hengst, U., Jaffrey, S. R. Function and translational regulation of mRNA in developing axons. Semin. Cell Dev. Biol. 18, 209-215 (2007).
- Deglincerti, A., Jaffrey, S. R. Insights into the roles of local translation from the axonal transcriptome. Open Biology. 2, 120079 (2012).
- Baleriola, J., et al. Axonally synthesized ATF4 transmits a neurodegenerative signal across brain regions. Cell. 158, 1159-1172 (2014).
- Gumy, L. F., et al. Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization. RNA. 17, 85-98 (2011).
- Taylor, A. M., et al. Axonal mRNA in uninjured and regenerating cortical mammalian axons. J. Neurosci. 29, 4697-4707 (2009).
- Yoon, B. C., et al. Local translation of extranuclear lamin B promotes axon maintenance. Cell. 148, 752-764 (2012).
- Dubacq, C., Jamet, S., Trembleau, A. Evidence for developmentally regulated local translation of odorant receptor mRNAs in the axons of olfactory sensory neurons. J. Neurosci. 29, 10184-10190 (2009).
- Willis, D. E., et al. Axonal Localization of transgene mRNA in mature PNS and CNS neurons. J. Neurosci. 31, 14481-14487 (2011).
- Kar, A. N., et al. Dysregulation of the axonal trafficking of nuclear-encoded mitochondrial mRNA alters neuronal mitochondrial activity and mouse. Dev. Neurobiol. 74, 333-350 (2014).
- Levsky, J. M., Singer, R. H. Fluorescence in situ hybridization: past, present and future. J. Cell Sci. 116, 2833-2838 (2003).
- Trcek, T., et al. Single-mRNA counting using fluorescent in situ hybridization in budding yeast. Nat. Protoc. 7, 408-419 (2012).
- Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods. 5, 877-879 (2008).
- Wang, F., et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J. Mol. Diagn. 14, 22-29 (2012).
- Ge, S., Crooks, G. M., McNamara, G., Wang, X. Fluorescent immunohistochemistry and in situ hybridization analysis of mouse pancreas using low-power antigen-retrieval technique. J. Histochem. Cytochem. 54, 843-847 (2006).
- Wang, H., et al. Dual-Color Ultrasensitive Bright-Field RNA In Situ Hybridization with RNAscope. Methods Mol. Biol. 1211, 139-149 (2014).
- Kluver, H., Barrera, E. A method for the combined staining of cells and fibers in the nervous system. J. Neuropathol. Exp. Neurol. 12, 400-403 (1953).
- Sheehan, D. C., Hrapchak, B. B. . Theory and Practice of Histotechnology. , (1987).
- Gross-Thebing, T., Paksa, A., Raz, E. Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos. BMC Biol. 12, 55 (2014).
- Farris, S., Lewandowski, G., Cox, C. D., Steward, O. Selective localization of arc mRNA in dendrites involves activity- and translation-dependent mRNA degradation. The Journal of neuroscience : the official journal of the Society for Neuroscience. 34, 4481-4493 (2014).
- Wang, F., et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. The Journal of molecular diagnostics : JMD. 14, 22-29 (2012).
- Sheehan, D. C., Hrapchak, B. B. . Theory and practice of histotechnology. , (1980).
