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。

注:所有程序符合美国国立卫生研究院指南实验动物的使用和护理。动物方案得到批准IACUC威尔康乃尔医学院。 1.组织准备<ol><li>颈椎脱位安乐死15定时女性怀孕。解剖胚胎E16 - 从定时怀孕的女性和地点胚胎E18胚胎逐个在6孔培养皿的孔中，填充有冷的磷酸盐缓冲盐水（PBS）。 </li><li>在冷PBS冲洗胚胎。 </li><li>在随后的基因分型的预制备的蛋白酶K溶液中取出胚胎和地点的羊膜的一小部分。或者，如果羊膜丢失，除去的胚胎尾巴尖的一小部分，并将其放置在蛋白酶K溶液</li><li>戳孔进入皮肤四周用昆虫针（至少100每边）的胚胎。 </li><li>删除PBS慢慢补充新鲜2％多聚甲醛（PFA），pH为7.4，向井。 </li><li>轻轻摇动2小时，4℃。 </li><li>在PBS中漂洗两次胚胎并在PBS中储存于4℃直至染色。 </li></ol> 2.基因分型<ol><li>根据制造商的说明浸入羊膜或胚胎尾巴在蛋白酶K混合物。 </li><li>孵育在56℃下进行10分钟。 </li><li>使用市售的DNA纯化试剂盒提取DNA。 </li><li>取0.5微升的总200微升纯化的基因组DNA溶液，用0.5微升每个基因特异性义和反义引物（10μM）的，2微升dNTP混合物结合它（2.5毫的dATP，dCTP，dGTP和dTTP的） ，0.2微升Taq聚合酶（5U /微升）和2微升的PCR缓冲液（10倍），加入的H 2 O来的20微升的总体积。 注:引物序列如下（ 见表1）: TrkA的:TrkA_F:GCG GGC GCC GCC GCG ATG，TrkA_R:GAA GCC GCC TGC GCG GCT CTG CCA GGG TG; PCR FRAG换货长度:400个碱基对（BP）。 Bax蛋白:IN5R:GTT GAC CAG AGT GGC GTA GG，Ex5F:TGA TCA GAA CCA TCA TG，NeoR:CCG CTT CCA TTG CTC AGC GG; PCR片段长度:野生型，304基点; Bax的空，507基点。 TauLacZ:lacZup:ACA ACG TCG TGA GTG GGA AAA，lacZdown:ATC AAC ATT AAA TGT GAG CGA G组; PCR片段长度:360基点。 </li><li>运行的PCR与TrkA的下列程序:步骤1:1分钟95℃，第2步:10秒，在95℃，第3步:20秒68℃，第4步:30秒72℃。 40次4 - 重复步骤2。 <ol><li>为的LacZ和Bax，运行的PCR使用以下程序:步骤1:在95℃下1分钟，步骤2:10秒，在95℃，第3步:1分钟在54℃，步骤4:1，在72分钟°C，重复步骤2 - 4 35个循环。 </li></ol></li><li>上，在100 V在1×Tris-醋酸盐-EDTA缓冲液的DNA梯的泳道2％琼脂糖凝胶进行20分钟运行的PCR样品，以评估片段长度。 </li><li>对于野生型的TrkA，T的存在分析胚胎RKA-tauLacZ和Bax无效等位基因。实验胚胎的基因型是TrkA的taulacZ / WT:Bax蛋白- / -和TrkA的taulacZ / taulacZ:Bax蛋白- / - ，取决于实验的问题。胚胎缺乏tauLacZ记者将负轴突染色，因此可以作为阴性对照来校准染色过程。 </li></ol> 3.胚胎染色<ol><li>使用商业套件的lacZ染色在3.2节下面介绍一些修改- 3.8。在这里，用微孔套件。 </li><li>沉浸在缓冲液A胚胎至少1小时，在室温下轻轻摇晃。 </li><li>直接浸入在缓冲液B的胚胎为至少15分钟，在室温下轻轻摇动。 </li><li>而胚胎是在缓冲液A / B中，在37℃下预暖组织中相应的解决方案。使用将5ml每胚胎组织中相应的解决方案。 </li><li>当胚胎在缓冲液A / B，准备新鲜的5-BRomo- 4-氯-3-吲哚基α-D-吡喃半乳糖苷（X-gal的）以40毫克/毫升在100％二甲基亚砜（DMSO）的浓度。 </li><li>稀X-gal的成以1mg / ml的浓度的预热组织基本溶液和再加5毫升X-gal的/组织中相应混合物到玻璃闪烁瓶中。 </li><li>转移的胚胎含有X-gal的/组织中相应混合物的玻璃闪烁管中。密封的盖子封口膜，37℃过夜，检查蓝染色存在。 </li><li> Postfix的胚胎与新鲜的4％PFA，pH值7.4。轻轻摇动4小时，4℃。 </li><li>在冷PBS漂洗胚胎两次。 </li></ol> 4.组织脱水及结算所<ol><li>为30分钟，在室温下，摇动在玻璃小瓶中发生的胚胎在50％甲醇/ 50％PBS中的混合物。 </li><li>继续在75％甲醇/ 25％PBS中脱水30分钟，在室温下摇动在玻璃小瓶中。 </li><li>继续在90％的脱水甲醇/ 10％的PBS 30分钟，在室温下摇动在玻璃小瓶中。 </li><li>继续在100％甲醇进行脱水30分钟（2次），在室温下摇动在玻璃小瓶中。 </li><li>而胚胎脱水，制备1:1（体积:体积）苯甲酰醇和苯甲酰苯甲酸的混合物。 注:苯甲酰醇和苯甲酰苯甲酸酯是有毒的。 </li><li>沉浸胚胎，在1:1（体积:体积）100％甲醇的混合物:100％BABB 15分钟。只使用玻璃，因为BABB会溶解塑料。 </li><li>胚胎沉浸在100％BABB彻底清除组织和可视化X-gal染色的清除胚胎。 注:尽快，因为X-gal的染色会褪色随着时间的推移图像。 </li></ol> 5.全山成像<ol><li>稳定的胚胎，沉浸在100％BABB，在适当的角度，使用摄影金属镊子。照亮使用的上行光并根据光的光源的组合。 </li><li>用解剖显微镜获取图像配备的数码相机。取多个明场曝光于五个连续焦平面相距0.5毫米沿Z轴。曝光时间和光圈值可以根据照明和摄像机的规格变化，并且需要为每个设置进行优化。 </li><li>使用标准的图像处理软件来处理的叠加图像，并生成合成照片。 </li></ol>

<ol>
	<li>Verze, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+innervation+in+hereditary+sensory+and+autonomic+neuropathy+type+IV.">Cutaneous innervation in hereditary sensory and autonomic neuropathy type IV.</a> Neurology. 55, 126-128 (2000).</li><li>Sethna, N. F., Meier, P. M., Zurakowski, D., Berde, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+sensory+abnormalities+in+children+and+adolescents+with+complex+regional+pain+syndromes.">Cutaneous sensory abnormalities in children and adolescents with complex regional pain syndromes.</a> Pain. 131, 153-161 (2007).</li><li>Uceyler, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+fibers+in+Fabry+disease:+baseline+and+follow-up+data+under+enzyme+replacement+therapy.">Small fibers in Fabry disease: baseline and follow-up data under enzyme replacement therapy.</a> J Peripher Nerv Syst. 16, 304-314 (2011).</li><li>Reichardt, L. F., Mobley, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+the+distance,+or+not,+with+neurotrophin+signals.">Going the distance, or not, with neurotrophin signals.</a> Cell. 118, 141-143 (2004).</li><li>White, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronous+onset+of+NGF+and+TrkA+survival+dependence+in+developing+dorsal+root+ganglia.">Synchronous onset of NGF and TrkA survival dependence in developing dorsal root ganglia.</a> J Neurosci. 16, 4662-4672 (1996).</li><li>Farinas, I., Wilkinson, G. A., Backus, C., Reichardt, L. F., Patapoutian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+neurotrophin+and+Trk+receptor+functions+in+developing+sensory+ganglia:+direct+NT-3+activation+of+TrkB+neurons+in+vivo.">Characterization of neurotrophin and Trk receptor functions in developing sensory ganglia: direct NT-3 activation of TrkB neurons in vivo.</a> Neuron. 21, 325-334 (1998).</li><li>Markus, A., Zhong, J., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raf+and+akt+mediate+distinct+aspects+of+sensory+axon+growth.">Raf and akt mediate distinct aspects of sensory axon growth.</a> Neuron. 35, 65-76 (2002).</li><li>Kuruvilla, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neurotrophin+signaling+cascade+coordinates+sympathetic+neuron+development+through+differential+control+of+TrkA+trafficking+and+retrograde+signaling.">A neurotrophin signaling cascade coordinates sympathetic neuron development through differential control of TrkA trafficking and retrograde signaling.</a> Cell. 118, 243-255 (2004).</li><li>Zhong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raf+kinase+signaling+functions+in+sensory+neuron+differentiation+and+axon+growth+in+vivo.">Raf kinase signaling functions in sensory neuron differentiation and axon growth in vivo.</a> Nature. 10, 598-607 (2007).</li><li>Bulfone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+olfactory+sensory+map+develops+in+the+absence+of+normal+projection+neurons+or+GABAergic+interneurons.">An olfactory sensory map develops in the absence of normal projection neurons or GABAergic interneurons.</a> Neuron. 21, 1273-1282 (1998).</li><li>Moqrich, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expressing+TrkC+from+the+TrkA+locus+causes+a+subset+of+dorsal+root+ganglia+neurons+to+switch+fate.">Expressing TrkC from the TrkA locus causes a subset of dorsal root ganglia neurons to switch fate.</a> Nature. 7, 812-818 (2004).</li><li>Knudson, C. M., Tung, K. S., Tourtellotte, W. G., Brown, G. A., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bax-deficient+mice+with+lymphoid+hyperplasia+and+male+germ+cell+death.">Bax-deficient mice with lymphoid hyperplasia and male germ cell death.</a> Science. 270 (5233), 96-99 (1995).</li><li>Lentz, S. I., Knudson, C. M., Korsmeyer, S. J., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+support+the+development+of+diverse+sensory+axon+morphologies.">Neurotrophins support the development of diverse sensory axon morphologies.</a> J. Neurosci. 19, 1038-1048 (1999).</li><li>Patel, T. D., Jackman, A., Rice, F. L., Kucera, J., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+sensory+neurons+in+the+absence+of+NGF/TrkA+signaling+in+vivo.">Development of sensory neurons in the absence of NGF/TrkA signaling in vivo.</a> Neuron. 25, 345-357 (2000).</li><li>Donovan, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-RAF+kinase+drives+developmental+axon+growth+and+promotes+axon+regeneration+in+the+injured+mature+CNS.">B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS.</a> The Journal of experimental medicine. 211, 801-814 (2014).</li><li>Mercer, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+endogenous+oncogenic+V600EB-raf+induces+proliferation+and+developmental+defects+in+mice+and+transformation+of+primary+fibroblasts.">Expression of endogenous oncogenic V600EB-raf induces proliferation and developmental defects in mice and transformation of primary fibroblasts.</a> Cancer research. 65, 11493-11500 (2005).</li><li>Tronche, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+glucocorticoid+receptor+gene+in+the+nervous+system+results+in+reduced+anxiety.">Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety.</a> Nature genetics. 23, 99-103 (1999).</li><li>Madisen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+toolbox+of+Cre-dependent+optogenetic+transgenic+mice+for+light-induced+activation+and+silencing.">A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing.</a> Nat Neurosci. 15, 793-802 (2012).</li><li>Feng, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Neuronal+Subsets+in+Transgenic+Mice+Expressing+Multiple+Spectral+Variants+of+GFP.">Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Schmidt, H., Rathjen, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DiI-labeling+of+DRG+neurons+to+study+axonal+branching+in+a+whole+mount+preparation+of+mouse+embryonic+spinal+cord.">DiI-labeling of DRG neurons to study axonal branching in a whole mount preparation of mouse embryonic spinal cord.</a> J Vis Exp. , (2011).</li></ol>

The authors declare that there are no competing interests.

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备组织一次段和评估生长轴突可能受影响的多种方式通过任何一项遗传操作的复杂的表型。该TrkA的taulacZ / taulacZ:Bax蛋白- / -行使一个基因的全面影响评估对周围伤害感受器轴突发展的兴趣。 该技术是仅限于伤害感受器轴突轨迹，在早期发育阶段通常会表达TrkA的可视化。除了外周伤害感受器，它可能显现交感神经和基底前脑胆碱能预测;然而这将需要的染色技术特定优化组织清算是这里所描述的协议的关键一步。它能够在全安装的准备更深躺在预测，因而不同的表型的潜在鉴定详细评估。荧光报告，如TdTomato或GFP的记者18,19，可以提供在特定条件下的视觉清晰度，然而，很难获得整个贴装图像，因为我们已经发现，在相对 ​​于在X-gal染色，荧光信号期间淡出基本上组织清算程序。 DiI标记染色也被广泛用于轴突标记20;其缺点是，它不能选择性地标记轴突的特定亚型，如伤害感受器纤维这里，并且难以在周边以充分标记长轨迹。 总之，我们提供了一个使用两个转基因小鼠系的组合，以允许在小鼠胚胎痛觉神经轴突的充分乔木的常规可视化的协议。

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。

<table><tbody><tr><td>PFA</td><td>Sigma-Aldrich</td><td>P6418</td><td></td></tr><tr><td>PBS</td><td>Life Tech</td><td>10010-023</td><td></td></tr><tr><td>Tissue Rinse Solution A</td><td>Millipore</td><td>BG-6-B</td><td></td></tr><tr><td>Tissue Rinse Solution B</td><td>Millipore</td><td>BG-7-B</td><td></td></tr><tr><td>Tissue Stain Base Solution</td><td>Millipore</td><td>BG-8-C</td><td></td></tr><tr><td>X-gal</td><td>Sigma-Aldrich</td><td>B4252</td><td></td></tr><tr><td>Glass scintiallation vial</td><td>Kimble Chase</td><td>74500-20</td><td></td></tr><tr><td>Incubator</td><td>Labline</td><td>Model 120</td><td></td></tr><tr><td>Insect pins</td><td>FST</td><td>26000-30</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>6-well dish</td><td>USA Scientific</td><td>CC7672-7506</td><td></td></tr><tr><td>Primers</td><td>IDT</td><td>custom DNA primers</td><td></td></tr><tr><td>Takara dNTP mixture</td><td>Takara</td><td>4030</td><td></td></tr><tr><td>Takara LA buffer</td><td>Takara</td><td>RR002A</td><td></td></tr><tr><td>Takara LA Taq</td><td>Takara</td><td>RR002A</td><td></td></tr><tr><td>PCR machine</td><td>Bio-Rad</td><td>DNA Engine Dyad</td><td></td></tr><tr><td>Benzyl alcohol</td><td>Sigma-Aldrich</td><td>B-1042</td><td></td></tr><tr><td>Benzyl benzoate</td><td>Sigma-Aldrich</td><td>B-6630</td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>M205A</td><td></td></tr><tr><td>Camera</td><td>Leica</td><td>DFC310FX</td><td></td></tr><tr><td>Ring light</td><td>Leica&amp;nbsp;</td><td>MEB110</td><td></td></tr><tr><td>Photoshop</td><td>Adobe</td><td>Photoshop 4.0</td><td></td></tr></tbody></table>

whole-mount imaging of mouse embryo sensory axon projections

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。

TrkA的WT / taulacZ的基因型:Bax蛋白- / -和TrkA的taulacZ / taulacZ:Bax蛋白- / -胚胎可以通过标准PCR基因分型来明确地确定（ 图1）。 X-gal染色显示在常规染色的胚胎（ 图2，图3a）详述周轴突乔木皮下，并在整个组织清除后的胚胎（ 图3b，4）。 我们已经孕育了TrkA的WT / taulacZ:Bax蛋白- / -与下神经元特异性巢子17控制携带组成激酶激活B-RAF（V600E）突变（LSL-kaBraf 16）自驾路线，获得LSL- kaBraf:TrkA的taulacZ / taulacZ:Bax蛋白- / -小鼠。在这些胚胎，这完全缺乏TrkA的信令，伤害感受器圆周突起的形态仍然开发了近正常（ 图4）。这证明对B-RAF的信令在外设轴突生长，并且还SA关键功能显示，存活和轴突生长促进的TrkA信令的功能可以被分离出来，与Bax蛋白- / -遗传背景代替的TrkA的依赖性存活信号的损失。 <img alt="图1" src="/files/ftp_upload/52212/52212fig1highres.jpg" /> 图1:转基因小鼠的基因分型代表琼脂糖凝胶图像。示例1:TrkA的WT / taulacZ:Bax的WT / - ，样品2:TrkA的taulacZ / taulacZ:Bax蛋白- / - ，样品3:TrkA的WT / taulacZ:Bax蛋白- / - 。 <img alt="图2" src="/files/ftp_upload/52212/52212fig2highres.jpg" /> 图2:E18.5的TrkA WT / taulacZ小鼠染色用X-gal从三叉神经节轴突突起在躯干和HEA。d的显示在一个未清除的胚胎。比例尺:1毫米。 <img alt="图3" src="/files/ftp_upload/52212/52212fig3highres.jpg" /> 图3:前E18.5 TrkA的WT / taulacZ鼠标染色用X-gal（A）及（B）与BABB从病种付费中旬躯干预测结算后显示。比例尺:2毫米。 <img alt="图4" src="/files/ftp_upload/52212/52212fig4highres.jpg" /> 图4:一个E16.5 LSL-kaBraf:TrkA的taulacZ / taulacZ:Bax蛋白- / - :巢蛋白的Cre小鼠胚胎染色用X-gal和清除与BABB kaBraf编码激酶活化的B-Raf激酶蛋白B-RAF V600E。 KAB-RAF的DRG神经元中的表达导致了全长，接近正常的轴突延长在没有TrkA的信令。比例尺:1毫米。欲了解更多的图像，包括控制，见文献。 15， 图2。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td colspan="2">用于PCR的基因分型的引物</td></tr><tr><td rowspan="3"> 1 </td><td> TrkA的</td><td> DNA片段大小:400基点</td></tr><tr><td> TrkA_F </td><td> GCG GGC GCC GCC GCG ATG </td></tr><tr><td> TrkA_R </td><td> GAA GCC GCC TGC GCG GCT CTG C​​CA GGG TG </td></tr><tr><td rowspan="4"> 2 </td><td> Bax基因</td><td> DNA片段大小:304 bp的（野生型），507基点（BAX空） </td></tr><tr><td> IN5R </td><td> GTT GAC CAG AGT GGC GTA GG </td></tr><tr><td> Ex5F </td><td> TGA TCA GAA CCA TCA TG </td></tr><tr><td> NeoR </td><td> CCG CTT CCA TTG CTC AGC GG </td></tr><tr><td rowspan="3"> 3 </td><td> TauLacZ </td><td> DNA片段大小:360基点</td></tr><tr><td> lacZup </td><td> ACA ACG TCG TGA GTG GGA AAA </td></tr><tr><td> lacZdown </td><td> ATC AAC ATT AAA TGT GAG CGA摹</td></TR&gt; </tbody></table> 表1:用于PCR的基因分型的引物。

Watch this Scientific Journal Video about Whole-mount Imaging of Mouse Embryo Sensory Axon Projections at JoVE.com

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

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.

小鼠胚胎感觉轴突预测整个安装成像

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

whole-mount-imaging-of-mouse-embryo-sensory-axon-projections

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

10.0K 次观看.  Weill Medical College of Cornell University. 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.

视频: Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

科研

神经科学

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

发育生物学

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

小鼠皮肤及其毛囊图案和感觉轴突形态分析中的应用平山成像

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

研究脊椎动物轴向伸长和分割的三维和四维可视化分析方法

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

胚胎运动神经元轴突投影模式的可视化<em&gt;果蝇</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

用光片荧光显微镜观察运动轴突导航和小鼠胚轴突树枝状的定量化

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

小鼠大脑皮层细胞组织的大尺度三维成像

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

可视化发育中的小鼠大脑中的神经元和神经胶质细胞动力学

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

作者聚焦：探索发育中的大脑中的细胞迁移和基因作用

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...