Hagiwara Soya, Tsuneishi Kazuhiro, Takada Naoya, Yasuda Kenji
Department of Pure and Applied Physics, Graduate School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku, Tokyo 169-8555, Japan.
Department of Physics, School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku, Tokyo 169-8555, Japan.
Gels. 2025 Aug 21;11(8):668. doi: 10.3390/gels11080668.
Axon polarization is a fundamental process in neuronal development, providing the structural basis for directional signaling in neural circuits. Precise control of axon specification is, thus, essential for the bottom-up construction of neuronal networks with defined architecture and connectivity. Although neurite length and elongation timing have both been implicated as determinants of axonal fate, their relative contributions have remained unresolved due to technical limitations in manipulating these factors independently in conventional culture systems. Here, we developed a constructive neuroengineering platform based on modifiable agarose gel microstructures that enables dynamic, in situ control of neurite outgrowth length and timing during neuronal cultivation. This approach allowed us to directly address whether axon polarization depends primarily on neurite length or the order of neurite extension. Using a single-neurite elongation paradigm, we quantitatively defined two length thresholds for axon specification: a critical length of 43.3 μm, corresponding to a 50% probability of axonal differentiation, and a definitive length of 95.4 μm, beyond which axonal fate was reliably established. In experiments involving simultaneous or sequential elongation of two neurites, we observed that neurite length-not elongation order-consistently predicted axonal identity, even when a second neurite was introduced after the first had already begun to grow. The presence of a competing neurite modestly elevated the effective critical length, suggesting inhibitory interactions that modulate length thresholds. These findings provide the first direct experimental confirmation that neurite length is the primary determinant of axon polarization and demonstrate the utility of constructive microfabrication approaches for dissecting fundamental principles of neuronal polarity. Our platform establishes a powerful experimental foundation for future efforts to achieve complete control over axon and dendrite orientation during the engineered construction of functional neuronal circuits.
轴突极化是神经元发育中的一个基本过程，为神经回路中的定向信号传递提供结构基础。因此，精确控制轴突特化对于自下而上构建具有特定结构和连接性的神经网络至关重要。尽管神经突长度和伸长时间都被认为是轴突命运的决定因素，但由于在传统培养系统中独立操纵这些因素存在技术限制，它们的相对贡献仍未得到解决。在这里，我们开发了一个基于可修饰琼脂糖凝胶微结构的建设性神经工程平台，该平台能够在神经元培养过程中动态、原位控制神经突生长的长度和时间。这种方法使我们能够直接解决轴突极化是否主要取决于神经突长度或神经突延伸顺序的问题。使用单神经突伸长范式，我们定量定义了轴突特化的两个长度阈值：临界长度为43.3μm，对应于轴突分化的50%概率；确定长度为95.4μm，超过此长度轴突命运得以可靠确立。在涉及两个神经突同时或顺序伸长的实验中，我们观察到神经突长度而非伸长顺序始终能预测轴突身份，即使在第一个神经突已经开始生长后再引入第二个神经突也是如此。存在竞争神经突会适度提高有效临界长度，表明存在调节长度阈值的抑制性相互作用。这些发现首次提供了直接的实验证据，证明神经突长度是轴突极化的主要决定因素，并展示了建设性微加工方法在剖析神经元极性基本原理方面的效用。我们的平台为未来在功能性神经元回路的工程构建过程中实现对轴突和树突方向的完全控制奠定了强大的实验基础。
