Wnt5a Regulates Midbrain Dopaminergic Axon Growth and Guidance

Brette D Blakely,Malcolm K Horne,Clare L Parish,Ernest Arenas,Maria L Macheda,Chathurini V Fernando,Steven A Stacker,Christopher R Bye,Branden Nelson

doi:10.1371/journal.pone.0018373

Abstract

During development, precise temporal and spatial gradients are responsible for guiding axons to their appropriate targets. Within the developing ventral midbrain (VM) the cues that guide dopaminergic (DA) axons to their forebrain targets remain to be fully elucidated. Wnts are morphogens that have been identified as axon guidance molecules. Several Wnts are expressed in the VM where they regulate the birth of DA neurons. Here, we describe that a precise temporo-spatial expression of Wnt5a accompanies the development of nigrostriatal projections by VM DA neurons. In mice at E11.5, Wnt5a is expressed in the VM where it was found to promote DA neurite and axonal growth in VM primary cultures. By E14.5, when DA axons are approaching their striatal target, Wnt5a causes DA neurite retraction in primary cultures. Co-culture of VM explants with Wnt5a-overexpressing cell aggregates revealed that Wnt5a is capable of repelling DA neurites. Antagonism experiments revealed that the effects of Wnt5a are mediated by the Frizzled receptors and by the small GTPase, Rac1 (a component of the non-canonical Wnt planar cell polarity pathway). Moreover, the effects were specific as they could be blocked by Wnt5a antibody, sFRPs and RYK-Fc. The importance of Wnt5a in DA axon morphogenesis was further verified in Wnt5a −/− mice, where fasciculation of the medial forebrain bundle (MFB) as well as the density of DA neurites in the MFB and striatal terminals were disrupted. Thus, our results identify a novel role of Wnt5a in DA axon growth and guidance.

Highlights

Dopamine (DA) neurons within the ventral midbrain (VM) project to the striatum and prefrontal cortex forming the nigrostriatal, mesocortical and mesolimbic pathways, which are important for motor and cognitive functions
Neurons and neurites as well as higher expression at the ventricular zone compared to the mantle zone. (G) Merged image of tyrosine hydroxylase (TH) and Wnt5 expression depicted in E and F. (H) At E14.5, during maturation of the midbrain dopamine pathways, a coronal section revealed Wnt5a expression was maintained in the VM, overlapping with (I) the TH+ cells. (J) Merged image of TH and Wnt5 expression. (K) Photomicrograph illustrating DA fibers in the medial forebrain bundle (MFB) approaching the lateral ganglionic eminence (LGE) at E14.5. (L–N) Sagittal section (medial to panel (K)) illustrating reversal of the Wnt5a gradient, with Wnt5a expression greater in the caudal VM than rostral VM. (M’) Enlargement from (M) illustrating dorsal trajectory of TH+ fibers (N’) Enlargement from (N) illustrating that Wnt5a is most likely secreted from TH2 cells
This is true for the dopaminergic pathways that arise from the ventral midbrain and innervate distant targets such as the striatum and cortex

Summary

Introduction

Dopamine (DA) neurons within the ventral midbrain (VM) project to the striatum and prefrontal cortex forming the nigrostriatal, mesocortical and mesolimbic pathways, which are important for motor and cognitive functions. Whilst the cues that orchestrate the birth of midbrain DA neurons are well established, the signals regulating DA neurite morphogenesis (including neurite growth, axon guidance and synaptogenesis) are less well defined. Several studies have identified cellular and molecular signals that participate in establishing these pathways (see review by [1]), including Ephrins [2,3,4], Semaphorins [5,6,7,8,9], Netrins and Slits [10,11], Engrailed-1 [12,13], and Sonic hedgehog [14]. In this study we asked whether Wnts regulate DA axon morphogenesis

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Results

Conclusion