Molecular Biology of Neural Development, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada; Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada; Program in Neuroengineering, McGill University, Montreal, Quebec, Canada.
Program in Neuroengineering, McGill University, Montreal, Quebec, Canada; Division of Engineering, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates.
PLoS Biol. 2015 Mar 31;13(3):e1002119. doi: 10.1371/journal.pbio.1002119. eCollection 2015 Mar.
During nervous system development, gradients of Sonic Hedgehog (Shh) and Netrin-1 attract growth cones of commissural axons toward the floor plate of the embryonic spinal cord. Mice defective for either Shh or Netrin-1 signaling have commissural axon guidance defects, suggesting that both Shh and Netrin-1 are required for correct axon guidance. However, how Shh and Netrin-1 collaborate to guide axons is not known. We first quantified the steepness of the Shh gradient in the spinal cord and found that it is mostly very shallow. We then developed an in vitro microfluidic guidance assay to simulate these shallow gradients. We found that axons of dissociated commissural neurons respond to steep but not shallow gradients of Shh or Netrin-1. However, when we presented axons with combined Shh and Netrin-1 gradients, they had heightened sensitivity to the guidance cues, turning in response to shallower gradients that were unable to guide axons when only one cue was present. Furthermore, these shallow gradients polarized growth cone Src-family kinase (SFK) activity only when Shh and Netrin-1 were combined, indicating that SFKs can integrate the two guidance cues. Together, our results indicate that Shh and Netrin-1 synergize to enable growth cones to sense shallow gradients in regions of the spinal cord where the steepness of a single guidance cue is insufficient to guide axons, and we identify a novel type of synergy that occurs when the steepness (and not the concentration) of a guidance cue is limiting.
在神经系统发育过程中，Sonic Hedgehog（Shh）和 Netrin-1 的梯度吸引着连合轴突的生长锥向胚胎脊髓的基板移动。Shh 或 Netrin-1 信号传导缺陷的小鼠存在连合轴突导向缺陷，这表明 Shh 和 Netrin-1 两者都需要正确的轴突导向。然而，Shh 和 Netrin-1 如何协作来引导轴突尚不清楚。我们首先量化了脊髓中 Shh 梯度的陡峭程度，发现它大多非常浅。然后，我们开发了一种体外微流控导向测定法来模拟这些浅梯度。我们发现，分离的连合神经元轴突对陡峭而不是浅的 Shh 或 Netrin-1 梯度有反应。然而，当我们将 Shh 和 Netrin-1 梯度结合在一起时，轴突对导向线索的敏感性增强，对仅存在一个线索时无法引导轴突的更浅梯度做出反应。此外，这些浅梯度仅在 Shh 和 Netrin-1 结合时极化生长锥 Src 家族激酶（SFK）活性，表明 SFK 可以整合这两个导向线索。总之，我们的研究结果表明，Shh 和 Netrin-1 协同作用使生长锥能够感知脊髓中单个导向线索的陡峭程度不足以引导轴突的浅梯度区域，并且我们确定了当导向线索的陡峭程度（而不是浓度）是限制因素时发生的一种新型协同作用。
