The role of tissue viscoelasticity in axon guidance during Xenopus brain development

Abstract

During nervous system development, axon bundles grow over large distances along well-defined pathways, guided by gradients of chemical cues in their environment. In addition, axons mechanically interact with their environment and respond to stiffness gradients they may encounter in the developing brain. In the model organism Xenopus laevis, retinal ganglion cell axons grow along the optic pathway toward the optic tectum. On their way through the mid-diencephalon, they turn caudally in response to gradients of the chemical guidance cues Semaphorin3A, Slit 1 and Slit 2 as well as tissue elasticity. However, while the importance of elasticity is becoming well-accepted in the context of development, biological tissues are not purely elastic but rather exhibit time-dependent mechanical properties known as viscoelasticity. In this project, I developed a method based on atomic force microscopy (AFM) to measure the viscoelasticity of biological tissues (in vivo Xenopus brain and ex vivo rodent spinal cord tissue). To investigate the influence of viscoelasticity on axon guidance, I first investigated the mechanical properties of the developing brain in vivo. I found that the Xenopus brain exhibited heterogeneous viscoelasticity and that greater tissue elasticity is correlated with a greater tissue viscosity. Subsequently, I wanted to understand how this heterogeneous tissue viscoelasticity may regulate axon growth. I developed tissue culture substrates with varying elasticities and viscosities and cultured Xenopus eye primordia explants on these substrates. I found that axons grew significantly longer on stiff elastic substrates compared to soft elastic substrates, reproducing previously published data, while there was no difference in axon length on soft and stiff viscoelastic substrates, indicating that viscosity is an important regulator of axon growth. I found that the regulation of axon length may be correlated with substrate timescale rather than by substrate elasticity. The substrate timescale is a measure of how long it takes a material to respond to an applied force or displacement; the shorter the timescale the faster the material response. Next, I used computer simulations to understand how the ratio of elastic and viscous tissue behaviour impacts axon growth. Exploiting the motor clutch model, which is well established for explaining the mechanical interactions of cells and their environment, I used physiological relevant mechanics I derived from the developing Xenopus brain and viscoelastic tissue culture substrates to simulate axon velocity, retrograde flow, and traction forces using elastic and viscoelastic frameworks. I found that the viscoelastic motor clutch model better described differences in axon lengths from the in vitro explant experiments. Furthermore, I found that the viscoelastic motor clutch model better describes axon velocities as a function of substrate timescale rather than the elastic framework. The characteristic substrate timescale appeared to be an important parameter in regulating axon growth. To further investigate if the viscoelastic motor clutch model was sufficient to explain axon growth, I investigated how traction forces and retrograde actin flow were regulated by substrate mechanics. To automate and simplify traction force microscopy (TFM) analysis, I developed custom software that enables users to perform TFM analysis with little prior understanding or knowledge. I found that growth cones mainly applied “pulling” forces to compliant substrates, and that the magnitude of the pulling force could be regulated by substrate timescale. Finally, I measured cytoskeletal dynamics of the growing axon and found that retrograde actin flow rates and microtubule polymerization rates were regulated by substrate mechanics. My results suggest that axon guidance is not only regulated by elasticity but also viscoelasticity and the viscous component should be considered when studying axon pathfinding. A better understanding of axon path finding could help us learn more about the role of mechanics in development and how to treat diseases in the central or peripheral nervous system.