Gradient steepness influences the pathfinding decisions of neuronal growth cones in vivo

Gradients of chemotropic molecules are generally thought to be fundamental for the guidance of neuronal growth cones in the developing embryo. Here we show that the grasshopper-secreted semaphorin Sema 2a is expressed in a gradient during the period of tibial Ti1 pioneer axon pathfinding into the CNS. At two critical Ti1 growth cone choice points, the Sema 2a gradient differs in steepness, whereas the absolute concentration is the same. Although Ti1 growth cones enter and extend up both steep and shallow gradients of Sema 2a, fewer projection errors occur along the steep gradient, suggesting that the steepness of the gradient encodes the critical guidance information into the pathfinding growth cone. In contrast, an increase in the absolute concentration of Sema 2a appears to constrain the Ti1 growth cone size. Using these in vivo gradients, we provide evidence that the Ti1 growth cone detects the Sema 2a gradient by measuring the fractional change in Sema 2a concentration, thereby demonstrating one mechanism that neuronal growth cones may use to detect and read gradients in vivo.