Imaging axon pathfinding in Xenopus in vivo

Axon pathfinding in the developing animal involves a highly dynamic process in which the axonal growth cone makes continuous decisions as it navigates toward its target. Changes occurring in the growth cone with respect to retracting from or extending into complex new territories can occur in minutes. Thus, the advent of strategies to visualize axon path-finding in vivo in a live intact animal is crucial for a better understanding of how the growth cone makes such rapid decisions in response to multiple cues. Combining these strategies with loss-of-function and/or gain-of-function techniques allows one to gain some insight as to which molecules are crucial to particular growth cone behaviors at specific choice points during navigation. The main advantage of using Xenopus lies in the accessibility of major axon tracts for live microscopy, as their embryonic development occurs ex utero. Furthermore, the robust embryos remain healthy during immobilization and allow for good imaging for long periods. This protocol describes the methods for stabilizing and preparing live Xenopus embryos for imaging labeled axonal tracts at high spatial and temporal resolution for up to 72 h. This approach can been used to investigate how the knockdown of certain gene functions can affect the speed of navigation through the well-studied Xenopus retinotectal pathway. It can be adapted to visualize other axon tracts of interest.