A new method to study nerve fiber projecting in the spinal cord during chicken embryo development

HU A-zhen1 YANG Ci-qing  FU SU-lei  JIA Yang-yang  LI Han  GUO Zhi-kun  LIN Jun-tang *

doi:10.3969/j.issn.0529-1356.2014.05.025

Acta Anatomica Sinica ›› 2014, Vol. 45 ›› Issue (5) : 724-728. DOI: 10.3969/j.issn.0529-1356.2014.05.025

A new method to study nerve fiber projecting in the spinal cord during chicken embryo development

HU A-zhen¹YANG Ci-qing ^1,2 FU SU-lei² JIA Yang-yang¹LI Han² GUO Zhi-kun¹LIN Jun-tang ^1,2*

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Abstract

Objective To develop a method of studying fiber projecting in the spinal cord duiring chicken embryo development. Methods At embryonic incubation 3 day (E3), pCAGGS-green fluorescent protein (GFP) plasmid was injected into the spinal cord usingin vivo electroporation. Three days after transfection (E6), GFP-positive embryos were collected under a stereo fluorescence microscope. Subsequently, the spinal cord was separated from the embryos and cut from the roof plate as an open book. After fixed with 4% paraformaldehyde (PFA) for one hour, the opened spinal cords were used for immunohistochemistry with N-cadherin antibody and with DAPI for nuclei. Finally, the nerve fiber projecting was photographed and analyzed under a fluorescence microscope. Results Based on the opened spinal cord and immunostaining in the cryosection, we observed that the nerve fibers projected across the midline of the floor plate and reached to the sulcus terminalis along the white matter of the contra side. The immunoreaction against N-cadherin indicated that overexpression of GFP has no significant effect on chicken embryonic development. Conclusion A new method to study fiber projecting in the developing chicken spinal cord is established successfully in this study.

Key words

Spinal cord / Nerve fiber / Projecting / Electroporationin vivo / Chicken embryo

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HU A-zhen1 YANG Ci-qing FU SU-lei JIA Yang-yang LI Han GUO Zhi-kun LIN Jun-tang *. A new method to study nerve fiber projecting in the spinal cord during chicken embryo development[J]. Acta Anatomica Sinica. 2014, 45(5): 724-728 https://doi.org/10.3969/j.issn.0529-1356.2014.05.025

References

［1］Kong WG, Wu XM, Zhang KN, et al. Differentiation of human bone marrow mesenchymal stem cells into motor neurons in vitro ［J］. Acta Academiae Medicinae Jiangxi, 2009,49(9): 1-4,139.(in Chinese)
孔卫国，吴晓敏，张昆南，等. 诱导人骨髓间充质干细胞向运动神经元分化的研究［J］. 江西医学院学报，2009，49（9）：1-4,139.
［2］Pan J，Huang ShM，Zhang L，et al. The afferent fibers project of rat cerebellum pompoms ［J］. Journal of Guangxi Medical University，2010，27（3）：368-369. (in Chinese)
潘剑，黄绍明，张亮，等. 大鼠小脑旁绒球的传入纤维投射［J］. 广西医科大学学报，2010，27（3）：368-369.
［3］Lois JH, Rice CD, Yates BJ. Neural circuits controlling diaphragm function in the cat revealed by transneuronal tracing ［J］. J Appl Physiol, 2009, 106(1):138-152.
［4］Yang L，Liu N，Yang LX, et al. Observation of connections between the fibers of the left and right spinal ganglion neurons with retrograde tracing technique of horseradish peroxidase ［J］. Chinese Journal of Clinical Rehabilitation，2005，9（41）：32-33. (in Chinese)
杨琳，刘娜，杨连雪，等. 应用辣根过氧化物酶逆行示踪技术观察左右侧脊神经节间纤维联系［J］. 中国临床康复，2005，9（41）：32-33.
［5］Reeber SL, Sakai N, Nakada Y, et al. Manipulating robo expression in vivo perturbs commissural axon pathfinding in the chick spinal cord［J］. J Neurosci, 2008, 28(35):8698-8708.
［6］Yang CQ, Shi XW, Hu AZh, et al.Observation of projections between the left and right spinal cord neuron fibers with electroporation GFP tracing technique in vivo of chick embryo ［J］. Chinese Journal of Immunology, 2012，28（8）：718-721. (in Chinese)
杨慈清，石晓卫，胡阿珍，等. 应用鸡胚活体电转GFP示踪技术观察脊髓左右两侧神经元纤维投射［J］.中国免疫学杂志，2012，28（8）：718-721. 
［7］Yang CQ, Mao HL, Guo ZhK, et al. Quality assessment of report gene green fluorescence protein in chicken embryo in vivo electroporation ［J］. Acta Anatomica Sinica，2012，43（4）：468-472. (in Chinese)
杨慈清，毛会丽，郭志坤，等.鸡胚活体原位电转基因技术报告基因绿色荧光蛋白质量评估［J］.解剖学报，2012，43（4）：468-472.
［8］Wang G, Li Y, Yang XS, et al.Gene regulation of cell migration during the development of early chick embryo ［J］. Chinese Journal of Cell Biology, 2011，33（10）：1069-1077. (in Chinese)
王广，李艳，杨雪松，等. 鸡胚早期发育过程中细胞迁移的基因调控［J］. 中国细胞生物学学报，2011，33（10）：1069-1077. 
［9］Andermatt I,Wilson N, Stoeckli ET. In ovo electroporation of miRNA-based-plasmids to investigate gene function in the developing neural tube ［J］.Methods Mol Biol, 2014,1101:353-368.
［10］Wilson NH, Stoeckli ET. In ovo electroporation of miRNA-based plasmids in the developing neural tube and assessment of phenotypes by Dil injection in open-book preparations［J］. J Vis Exp, 2012, (68).
［11］Sakai N, Insolera R, Sillitoe RV, et al. Axon sorting within the spinal cord marginal zone via Robo-mediated inhibition of N-cadherin controls spinocerebellar tract formation［J］. J Neurosci, 2012, 32(44):15377-15387.
［12］Phan KD, Butler SJ. Bilaterally symmetric populations of chicken dI1 (commissural) axons cross the floor plate independently of each other［J］. PLoS One,2013, 8(4): e62977.〖ZK)〗