Assaying the ability of diffusible signaling molecules to reorient embryonic spinal commissural axons.
Assaying the ability of diffusible signaling molecules to reorient embryonic spinal commissural axons.
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DOI：
10.3791/1853
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发表时间：
2010-03-08
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影响因子：
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通讯作者：
Butler, Samantha J
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作者：
Hazen, Virginia M;Phan, Keith;Butler, Samantha J
脊椎动物脊髓的背侧互交轴突(1)已成为识别轴突引导信号的宝贵模型系统。在这里，我们描述了一种体外实验，即“重定向实验”，该实验已被广泛用于研究外在和内在信号对交联轴突定向的影响(2)。这个实验是由Jane Dodd， Thomas Jessell和Andrew Lumsden实验室的许多人开发的（详见致谢），该实验的不同版本被用来证明关键轴突引导分子的重新定向活动，包括顶板中的BMP化学驱素（3,4）和脊髓底板中的Netrin1(5)和Sonic Hedgehog(6)的化学吸引活动。从胚胎日(E) 11大鼠中剥离2-3段脊髓背侧2 / 3的外植体，并在三维胶原凝胶中培养(7)。E11背侧脊柱外植体含有新生的联合神经元，可以通过其轴突糖蛋白Tag1的表达来识别(8)。在30-40小时的培养过程中，这些背侧外植体的互交轴突轨迹与在体内观察到的时间过程相似。这种轴突轨迹可以通过放置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外侧边缘接触来挑战。延伸到附加组织附近的共交轴突在内源性顶板和异位外侧组织信号的影响下生长。在这种情况下，互交轴突重新定向的程度是可以量化的。使用这种方法，既可以检查特定信号重新定向交轴突的充分性（3,4），也可以检查该信号指导交轴突轨迹的必要性(9)。
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).