Using genetic markers to establish in vivo roles for Robo receptors in commissural axon guidance within the chick spinal cord

Abstract

As axons grow in the developing embryo, they encounter intermediate targets along the way that break up their journey into short segments, which they navigate sequentially to ultimately reach their synaptic targets. These intermediate targets produce both attractants and repellents that push and pull, respectively, axons along each step of their route. The ventral midline (VM), in both vertebrates and invertebrates, is a key intermediate target or choice point for pathfinding axons. Commissural neurons are a well-studied system for investigating the mechanisms that regulate axon guidance at the VM. While pre-crossing axons travel along a relatively simple trajectory, post-crossing axons extend along a variety of complex trajectories on the contralateral side of the floor plate (FP). Focal applications of DiI recently identified that most post-crossing commissural axons turn rostrally into the longitudinal plane and either project alongside the FP (Medial Longitudinal Commissural; MLC) or grow into an intermediate region of the spinal cord (Intermediate Longitudinal Commissural; ILC). Whereas this approach provides unobstructed views of post-crossing trajectories, it cannot be used to reproducibly mark the same subsets of axons in each embryo. In chapter 1 of this thesis, I employ unilateral in ovo electroporation to show that murine Atoh1, Neurog1, and Neurog2 enhancers direct reporter gene expression to dI1 and dI2 commissural neurons/axons in the avian spinal cord, and that dI1 and dI2 axons elaborate rostrally-directed MLC and ILC trajectories on the contralateral side of the FP. In chapter 2 and 3, I then use this labeling system to assess the consequences of manipulating the expression of the repellent guidance receptors, Robo1 and Robo2 (Robo1/2), on these axons in vivo. In response to misexpressing a cytoplasmic truncation of Robo1/2, which should block all Robo-ligand interactions, post-crossing commissural axons extend alongside, but do not project away from or re-enter the FP. On the other hand, mis-expression of full length Robo2 prevents many commissural axons from crossing the VM. Together, these findings support key and selective in vivo roles for Robo receptors in altering the responsiveness of decussated commissural axons and promoting their expulsion from the VM within the chick spinal cord.