小鼠胚胎感觉轴突预测整个安装成像
Summary
We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.
Abstract
全长神经元的预测在胚胎的可视化是必须获得的神经网络如何发展哺乳动物的认识。在这里，我们描述的方法原位标记背根神经节（DRG）轴突预测的一个子集，使用多个基因操纵鼠标线，以评估他们的表型特征。所述的TrkA阳性神经元是伤害性感受器神经元，专门用于疼痛信号的传输。我们利用的TrkA taulacZ鼠标线标记所有的TrkA阳性轴突末梢的轨迹在完整的小鼠胚胎。我们进一步繁殖的TrkA taulacZ线到Bax缺无的背景，这基本上废除神经细胞凋亡，以评估的独立地对神经元存活的遗传操作的可能影响生长相关的问题。随后，感兴趣的转基因小鼠繁殖与TrkA的taulACZ / Bax的零线和然后准备用于使用本文所描述的技术的研究。此报告包括解剖，组织准备，染色和结算的时间对小鼠繁殖计划的详细信息，基因分型，以便在整个安装准备全长轴突轨迹可视化。
Introduction
建立精确的神经网络是一个复杂的发育过程中神经系统的功能是必不可少的。干扰在这个过程导致神经元功能障碍已与人类神经系统疾病1-3。研究轴突生长和靶神经支配的哺乳动物的分子机制，我们开发了一个协议，以可视化的TrkA表达感觉神经元使用两种转基因小鼠系的组合的轴突轨迹。
TrkA的是受体对神经生长因子NGF和是伤害性感觉神经元4的功能标记。早期发育过程中的TrkA高度表达在伤害感受神经元，并介导NGF依赖性神经元存活，轴突生长，树枝状和靶神经支配5-9。在的TrkA taulacZ小鼠中，野生型的TrkA基因置换为taulacZ表达式c磁带式10中 ，以使得推定的TrkA阳性神经元的轴突形态可通过β半乳糖苷酶（X-gal的）染色11可视化。使用杂的TrkA taulacZ / WT线，我们可以检查可调节或干扰体内感觉传入预测的发展的因素。
此外，TrkA的表达是不存在于纯合的TrkA taulacZ / taulacZ小鼠，这因此可以用来评估轴突生长促进机制在不存在NGF / TrkA的信令。由于疼痛神经元依赖于NGF / TrkA的信号不仅对轴突的生长，同时也为求生存，我们另外聘请鼠标线，缺乏促凋亡蛋白Bax基因，抑制细胞凋亡在胚胎DRG神经元细胞死亡是另有解救出来在不存在的TrkA信令的观察。的Bax蛋白– / –背景12从而允许molecu信号通路专门影响轴突生长7-9,13-15的LAR解剖。在的TrkA – / – ：Bax蛋白– / –小鼠，DRG神经元存活，但在皮肤感觉传入神经 支配被完全取消14,15。我们可以有选择地激活的信号通路，以确定它们各自的轴突预测的发展作出了贡献。该方法的效用是，它允许改变在轴突生长表型的评估当不同的遗传修饰的繁殖到的TrkA taulacZ / taulacZ：Bax蛋白– / –或TrkA的taulacZ / WT：Bax蛋白– / –背景。
Protocol
Representative Results
Discussion
胚胎的TrkA taulacZ小鼠的上述X-gal染色过程允许长距离轴突突起，在完好的固定胚胎的快速和详细可视化。因为Bax缺无背景这些小鼠允许信令可能有助于既轴突生长和神经元存活机制的探测。有兴趣的转基因或基因敲除小鼠交配允许轴突表型的综合评估，可以服务于未来的实验作为有用的指导，研究轴突生长的信号更详细的方式。在体内轴突生长研究的一个主要挑战是准…
Disclosures
The authors have nothing to disclose.
Acknowledgements
笔者想感谢路易斯·雷查德博士为TrkA的taulacZ小鼠和安妮特·马库斯博士有见地的讨论和建议。这项工作是从伯克基金会以及白厅基金研究资助2010-08-61，从翅膀生命基金会（WFL-US-028/14）研究经费支持的启动资金，从授予ZB1-1102-1克里斯托弗＆达娜里夫基金会和赠款1R01EY022409和3R01EY022409-01S1从国家眼科研究所，为JZ。 KJO是史密斯的家伙。
Materials
| Company | Catalog Number | |
| PFA | Sigma-Aldrich | P6418 |
| PBS | Life Tech | 10010-023 |
| Tissue Rinse Solution A | Millipore | BG-6-B |
| Tissue Rinse Solution B | Millipore | BG-7-B |
| Tissue Stain Base Solution | Millipore | BG-8-C |
| X-gal | Sigma-Aldrich | B4252 |
| Glass scintiallation vial | Kimble Chase | 74500-20 |
| Incubator | Labline | Model 120 |
| Insect pins | FST | 26000-30 |
| DMSO | Sigma-Aldrich | D8418 |
| 6 well dish | USA Scientific | CC7672-7506 |
| Primers | IDT | custom DNA primers |
| Takara dNTP mixture | Takara | 4030 |
| Takara LA buffer | Takara | RR002A |
| Takara LA Taq | Takara | RR002A |
| PCR machine | Bio-Rad | DNA Engine Dyad |
| Benzyl alcohol | Sigma-Aldrich | B-1042 |
| Benzyl benzoate | Sigma-Aldrich | B-6630 |
| Dissecting microscope | Leica | M205A |
| Camera | Leica | DFC310FX |
| Ring light | Leica | MEB110 |
| Photoshop | Adobe | Photoshop 4.0 |
References
- Verze, L., et al. Cutaneous innervation in hereditary sensory and autonomic neuropathy type IV. Neurology. 55, 126-128 (2000).
- Sethna, N. F., Meier, P. M., Zurakowski, D., Berde, C. B. Cutaneous sensory abnormalities in children and adolescents with complex regional pain syndromes. Pain. 131, 153-161 (2007).
- Uceyler, N., et al. Small fibers in Fabry disease: baseline and follow-up data under enzyme replacement therapy. J Peripher Nerv Syst. 16, 304-314 (2011).
- Reichardt, L. F., Mobley, W. C. Going the distance, or not, with neurotrophin signals. Cell. 118, 141-143 (2004).
- White, F. A., et al. Synchronous onset of NGF and TrkA survival dependence in developing dorsal root ganglia. J Neurosci. 16, 4662-4672 (1996).
- Farinas, I., Wilkinson, G. A., Backus, C., Reichardt, L. F., Patapoutian, A. Characterization of neurotrophin and Trk receptor functions in developing sensory ganglia: direct NT-3 activation of TrkB neurons in vivo. Neuron. 21, 325-334 (1998).
- Markus, A., Zhong, J., Snider, W. D. Raf and akt mediate distinct aspects of sensory axon growth. Neuron. 35, 65-76 (2002).
- Kuruvilla, R., et al. A neurotrophin signaling cascade coordinates sympathetic neuron development through differential control of TrkA trafficking and retrograde signaling. Cell. 118, 243-255 (2004).
- Zhong, J., et al. Raf kinase signaling functions in sensory neuron differentiation and axon growth in vivo. Nature. 10, 598-607 (2007).
- Bulfone, A., et al. An olfactory sensory map develops in the absence of normal projection neurons or GABAergic interneurons. Neuron. 21, 1273-1282 (1998).
- Moqrich, A., et al. Expressing TrkC from the TrkA locus causes a subset of dorsal root ganglia neurons to switch fate. Nature. 7, 812-818 (2004).
- Knudson, C. M., Tung, K. S., Tourtellotte, W. G., Brown, G. A., Korsmeyer, S. J. Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science. 270 (5233), 96-99 (1995).
- Lentz, S. I., Knudson, C. M., Korsmeyer, S. J., Snider, W. D. Neurotrophins support the development of diverse sensory axon morphologies. J. Neurosci. 19, 1038-1048 (1999).
- Patel, T. D., Jackman, A., Rice, F. L., Kucera, J., Snider, W. D. Development of sensory neurons in the absence of NGF/TrkA signaling in vivo. Neuron. 25, 345-357 (2000).
- Donovan, K. J., et al. B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS. The Journal of experimental medicine. 211, 801-814 (2014).
- Mercer, K., et al. Expression of endogenous oncogenic V600EB-raf induces proliferation and developmental defects in mice and transformation of primary fibroblasts. Cancer research. 65, 11493-11500 (2005).
- Tronche, F., et al. Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nature genetics. 23, 99-103 (1999).
- Madisen, L., et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nat Neurosci. 15, 793-802 (2012).
- Feng, G., et al. Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP. Neuron. 28, 41-51 (2000).
- Schmidt, H., Rathjen, F. G. DiI-labeling of DRG neurons to study axonal branching in a whole mount preparation of mouse embryonic spinal cord. J Vis Exp. , (2011).
