Hazen Virginia M, Phan Keith, Yamauchi Ken, Butler Samantha J
Department of Biological Sciences, University of Southern California, CA, USA.
J Vis Exp. 2010 Mar 8(37):1853. doi: 10.3791/1853.
Dorsal commissural axons in the vertebrate spinal cord(1) have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons(2). This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate(3,4) and the chemoattractive activities of Netrin1(5) and Sonic Hedgehog (Shh)(6) in the floor plate in the spinal cord. Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels(7). E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag1(8). Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons(3,4) as well the necessity for this signal to direct the commissural trajectory(9).
脊椎动物脊髓中的背侧连合轴突（1）一直是用于识别轴突导向信号的极有价值的模型系统。在此，我们描述一种体外实验方法，即“重定向实验”，该方法已被广泛用于研究外在和内在信号对连合轴突定向的影响（2）。此实验方法由简·多德、托马斯·杰塞尔和安德鲁·拉姆斯登实验室的众多人员共同开发（更多细节见致谢部分），该实验方法的不同版本被用于证明关键轴突导向分子的重定向活性，包括顶板中的骨形态发生蛋白（BMP）化学排斥分子（3,4）以及脊髓底板中Netrin1（5）和音猬因子（Shh）（6）的化学吸引活性。从胚胎第11天（E11）的大鼠中解剖出包含脊髓背侧三分之二2 - 3节段的外植体，并在三维胶原凝胶中培养（7）。E11背侧脊髓外植体包含新生的连合神经元，这些神经元可通过其轴突中糖蛋白Tag1的表达来识别（8）。在培养30 - 40小时的过程中，这些背侧外植体中连合轴突的轨迹得以重现，其时间进程与在体内观察到的相似。通过将测试组织或表达候选信号分子的COS细胞聚集体与背侧外植体的一侧边缘接触，可以对这种轴突轨迹进行挑战。在附加组织附近延伸的连合轴突将在内源性顶板和异位侧方组织信号的共同影响下生长。在这些情况下连合轴突重定向的程度可以进行量化。使用此实验方法，既可以检验特定信号使连合轴突重定向的充分性（3,4），也可以检验该信号对引导连合轴突轨迹的必要性（9）。
