Abstract
During embryogenesis motor axons navigate to their target muscles, where individual motor axons develop complex branch morphologies. The mechanisms that control axonal branching morphogenesis have been studied intensively, yet it still remains unclear when branches begin to form or how branch locations are determined. Live cell imaging of individual zebrafish motor axons reveals that the first axonal branches are generated at the ventral extent of the myotome via bifurcation of the growth cone. Subsequent branches are generated by collateral branching restricted to their synaptic target field along the distal portion of the axon. This precisely timed and spatially restricted branching process is disrupted in turnout mutants we identified in a forward genetic screen. Molecular genetic mapping positioned the turnout mutation within a 300 kb region encompassing eight annotated genes, however sequence analysis of all eight open reading frames failed to unambiguously identify the turnout mutation. Chimeric analysis and single cell labeling reveal that turnout function is required cell non-autonomously for intraspinal motor axon guidance and peripheral branch formation. turnout mutant motor axons form the first branch on time via growth cone bifurcation, but unlike wild-type they form collateral branches precociously, when the growth cone is still navigating towards the ventral myotome. These precocious collateral branches emerge along the proximal region of the axon shaft typically devoid of branches, and they develop into stable, permanent branches. Furthermore, we find that null mutants of the guidance receptor plexin A3 display identical motor axon branching defects, and time lapse analysis reveals that precocious branch formation in turnout and plexin A3 mutants is due to increased stability of otherwise short-lived axonal protrusions. Thus, plexin A3 dependent intrinsic and turnout dependent extrinsic mechanisms suppress collateral branch morphogenesis by destabilizing membrane protrusions before the growth cone completes navigation into the synaptic target field.
Highlights
IntroductionMotor axons navigate to their muscle targets where they generate elaborate axonal branches that synapse on multiple muscle fibers [1,2]
During vertebrate development, motor axons navigate to their muscle targets where they generate elaborate axonal branches that synapse on multiple muscle fibers [1,2]
Taking advantage of its amenability for genetic approaches and live cell imaging of individual neurons, we focus here on an identified motor neuron, Caudal Primary (CaP), to identify genes and mechanism that regulate its intricate axonal branching pattern
Summary
Motor axons navigate to their muscle targets where they generate elaborate axonal branches that synapse on multiple muscle fibers [1,2]. At the cellular level, branching begins with the localized accumulation of actin, and the de-bundling of microtubules at the nascent branch point, followed by the formation and extension of f-actin rich membrane protrusions into which microtubules subsequently extend [8,9]. While microtubules stabilize axonal protrusions, several intracellular regulators have been implicated in the transition from axon protrusions into stable branches These factors include the C. elegans ubiquitin ligase Rpm-1 [10,11,12], and the phosphatidylinositol 3-kinase (PI3K)/protein kinases AKT/ glycogen synthase kinase 3 (GSK3) pathway in dorsal root ganglia neurons [10,11]
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