Functional Characterization of Netrin-1 and Its Role in CNS-PNS Boundary Maintenance in the Developing Vertebrate Brainstem

Abstract

Netrin-1 (Ntn1) has represented the archetype of diffusible guidance cues since its discovery in the 1990s. It is often depicted in a gradient emanating from the floor plate of the embryonic neural tube, through which it instructs ventral commissural axon guidance. However, Ntn1 can also act permissively and over short-range. The molecular basis for Ntn1’s functional versatility is unknown for two major reasons. First, because until recently the only available mutant alleles of Ntn1 were severely hypomorphic, the full extent of Ntn1’s functions in vivo remained unclear, as the absence of phenotypes could reflect either the activity of alternative ligands or residual Ntn1. Second, there are few systems where Ntn1’s permissive and instructive activities can be clearly distinguished. To address these challenges, I defined the scope of Ntn1’s activities in vivo using a new Ntn1 null allele. By analyzing tissues in Ntn1 null mice that exhibit phenotypic discrepancies with Ntn1 receptor mutants and Ntn1 hypomorphs, I showed that low levels of Ntn1 account for persistent commissural attraction to the midline in the hypomorphs. In contrast, Ntn1 is not necessarily the dominant ligand for Unc5 family members in vivo and may not play a major role in survival or angiogenesis. Using the Ntn1 null mice, I also discovered a new local and permissive role for Ntn1 in the vertebrate brainstem. During development, rhombic lip-derived neurons undertake extensive tangential migrations throughout the hindbrain; many migrate near cranial nerves yet remain in the CNS. I found that Ntn1 accumulates beneath the pial surface separating the CNS from the PNS, with gaps of protein at nerve roots. Loss of Ntn1 from the sub-pial region causes hindbrain neurons to enter cranial nerves and enter the periphery; conversely, expression of Ntn1 throughout the mutant hindbrain can prevent their departure. We propose that Ntn1 confines rhombic lip-derived neurons by providing a preferred substrate for tangentially migrating neurons in the SPR, preventing their entry into nerve roots. Since Ntn1 plays distinct roles in the confinement and guidance of rhombic lip neurons, this model system provides an opportunity to dissect the mechanism underlying different modes of Ntn1 function.