Department of Life Science and Biotechnology, Tokyo University of Agriculture and Technology, Tokyo, Japan.
J Neurochem. 2012 Dec;123(6):904-10. doi: 10.1111/jnc.12001. Epub 2012 Oct 4.
Formation of an axon is the first morphological evidence of neuronal polarization, visible as a profound outgrowth of the axon compared with sibling neurites. One unsolved question on the mechanism of axon formation is the role of axon outgrowth in axon specification. This question was difficult to assess, because neurons freely extend their neurites in a conventional culture. Here, we leveraged surface nano/micro-modification techniques to fabricate a template substrate for constraining neurite lengths of cultured neurons. Using the template, we asked (i) Do neurons polarize even if all neurites cannot grow sufficiently long? (ii) Would the neurite be fated to become an axon if only one was allowed to grow long? A pattern with symmetrical short paths (20 μm) was used to address the former question, and an asymmetrical pattern with one path extended to 100 μm for the latter. Axon formation was evaluated by tau-1/MAP2 immunostaining and live-cell imaging of constitutively-active kinesin-1. We found that (1) neurons cannot polarize when extension of all neurites is restricted and that (2) when only a single neurite is permitted to grow long, neurons polarize and the longest neurite becomes the axon. These results provide clear evidence that axon outgrowth is required for its specification.
轴突的形成是神经元极化的第一个形态学证据，与兄弟姐妹的神经突相比，轴突明显向外生长。轴突形成机制中一个未解决的问题是轴突生长在轴突特化中的作用。这个问题很难评估，因为神经元在常规培养中可以自由地延伸它们的神经突。在这里，我们利用表面纳米/微加工技术来制造一个模板基底，以限制培养神经元的神经突长度。使用该模板，我们提出了以下两个问题：（i）即使所有的神经突都不能充分生长，神经元是否会极化？（ii）如果只允许一个神经突生长得足够长，那么这个神经突是否会注定成为轴突？采用对称的短路径（20 μm）图案来解决第一个问题，而采用不对称的图案，其中一个路径延伸到 100 μm，用于解决第二个问题。通过 tau-1/MAP2 免疫染色和组成型激活的驱动蛋白-1 的活细胞成像来评估轴突的形成。我们发现：（1）当所有神经突的延伸都受到限制时，神经元不能极化；（2）当只有一个神经突被允许生长得足够长时，神经元会极化，最长的神经突成为轴突。这些结果提供了明确的证据，表明轴突生长是其特化所必需的。
