Wnts guide longitudinal axon tracts in the brain

Abstract

The human brain contains more than 10 billion neurons that form over 10 trillion connections. The establishment of these connections during development requires axons to extend through the extracellular environment to their synaptic targets. This process of axon guidance is mediated by molecular cues in the extracellular matrix known as axon guidance molecules. Longitudinal axon tracts such as the striatonigral (SN), striatopallidal (SP), dopaminergic (mdDA) and serotonergic (5HT) pathways require molecular signals for their proper development. Impairment in their connectivity is implicated in Huntington’s diseases, Parkinson’s disease, drug addiction, depression and schizophrenia. Surprisingly little is known about the molecular events that underlie the establishment of these pathways. Wnt proteins are secreted molecules generally expressed in gradients along the anterior-posterior (A-P) axis of the developing nervous system inducing axon growth and guidance by binding axonal receptors such as Frizzleds and Ryk. This thesis explores a novel role for Wnts and their receptors in the A-P guidance of SP, SN, mdDA and 5HT axon tracts and for the first time establishes a role for Wnt/PCP signalling in the formation of these axon tracts. The SP and the SN axon tracts originate from the striatum and project to the substantia nigra retiulate (SNr) indirectly or directly, respectively. Our results show that SP and the SN axons grow alongside to the GP and that during this early phase of axon guidance the Frizzled3 (Fzd3) receptor is required for GP entry. Our findings are the first to demonstrate that during specific parts of their trajectories SP and SN axons use common molecular cues for navigating target structures such as the GP. mdDA axons grow in the rostral direction to form axon projections in the forebrain. We discovered that Wnt/PCP signalling dictates the rostral orientation of mdDA axons at the level of the midbrain. The core PCP receptors Fzd3, Vangl2 and Celsr3 were expressed in mdDA neurons and axons. Additionally, Wnt5a and Wnt7b were expressed in opposite gradients in the midbrain. Using collagen assays, we demonstrated that Wnt5a is a chemorepellent and Wnt7b a chemoattractant cue for mdDA axons. Serotonergic (5HT) neurons send their ascending axons to the forebrain and descending axons project to the spinal. 5HT neurons are born in the ventricular zone and migrate to the raphe by somal translocation. We found that for 5HT neurons, axon guidance appears to precede the final orientation of the soma. In wild-type animals, cell bodies of migrating, descending 5HT neurons initially point laterally but begin to reorient along the A–P axis as they approach their final lateral position, thereby obeying the direction of their axon projections. Our open book assay shows that the 5HT axons are responsive to Wnt4, Wnt5a and Wnt7a. Furthermore, they require core PCP receptors such as Fzd3, Celsr3 and Vangl2 to sense the Wnts. Overall our findings not only help to understand how these longitudinal axon connections are established but also provide a framework for understanding how these pathways may be changed in disease and could be altered in therapeutic settings.