Dataset Information

Pioneer midbrain longitudinal axons navigate using a balance of Netrin attraction and Slit repulsion.

ABSTRACT:

Background

Longitudinal axons grow parallel to the embryonic midline to connect distant regions of the central nervous system. Previous studies suggested that repulsive midline signals guide pioneer longitudinal axons by blocking their entry into the floor plate; however, the role of midline attractants, and whether attractant signals may cooperate with repulsive signals, remains unclear. In this study we investigated the navigation of a set of pioneer longitudinal axons, the medial longitudinal fasciculus, in mouse embryos mutant for the Netrin/Deleted in Colorectal Cancer (DCC) attractants, and for Slit repellents, as well as the responses of explanted longitudinal axons in vitro.

Results

In mutants for Netrin1 chemoattractant or DCC receptor signaling, longitudinal axons shifted away from the ventral midline, suggesting that Netrin1/DCC signals act attractively to pull axons ventrally. Analysis of mutants in the three Slit genes, including Slit1/2/3 triple mutants, suggest that concurrent repulsive Slit/Robo signals push pioneer axons away from the ventral midline. Combinations of mutations between the Netrin and Slit guidance systems provided genetic evidence that the attractive and repulsive signals balance against each other. This balance is demonstrated in vitro using explant culture, finding that the cues can act directly on longitudinal axons. The explants also reveal an unexpected synergy of Netrin1 and Slit2 that promotes outgrowth.

Conclusions

These results support a mechanism in which longitudinal trajectories are positioned by a push-pull balance between opposing Netrin and Slit signals. Our evidence suggests that longitudinal axons respond directly and simultaneously to both attractants and repellents, and that the combined signals constrain axons to grow longitudinally.

SUBMITTER: Kim M

PROVIDER: S-EPMC4118263 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Publications

Pioneer midbrain longitudinal axons navigate using a balance of Netrin attraction and Slit repulsion.

Kim Minkyung M Farmer W Todd WT Bjorke Brielle B McMahon Samuel A SA Fabre Pierre J PJ Charron Frédéric F Mastick Grant S GS

Neural development 20140724

<h4>Background</h4>Longitudinal axons grow parallel to the embryonic midline to connect distant regions of the central nervous system. Previous studies suggested that repulsive midline signals guide pioneer longitudinal axons by blocking their entry into the floor plate; however, the role of midline attractants, and whether attractant signals may cooperate with repulsive signals, remains unclear. In this study we investigated the navigation of a set of pioneer longitudinal axons, the medial long ...[more]

PMID: 25056828

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Project description:Modulation of microtubule (MT) dynamics is a key event of cytoskeleton remodeling in the growth cone (GC) during axon outgrowth and pathfinding. Our previous studies have shown that the direct interaction of netrin receptor DCC and DSCAM with polymerized TUBB3, a neuron-specific MT subunit in the brain, is required for netrin-1-mediated axon outgrowth, branching, and attraction. Here, we show that uncoupling of polymerized TUBB3 with netrin-1-repulsive receptor UNC5C is involved in netrin-1-mediated axonal repulsion. TUBB3 directly interacted with UNC5C and partially colocalized with UNC5C in the peripheral area of the GC of primary neurons from the cerebellar external granule layer of P2 mouse pups of both sexes. Netrin-1 reduced this interaction as well as the colocalization of UNC5C and TUBB3 in the GC. Results from the in vitro cosedimentation assay indicated that UNC5C interacted with polymerized TUBB3 in MTs and netrin-1 decreased this interaction. Knockdown of either TUBB3 or UNC5C blocked netrin-1-promoted axon repulsion in vitro and caused defects in axon projection of DRG toward the spinal cord in vivo Furthermore, live-cell imaging of end-binding protein 3 tagged with EGFP (EB3-GFP) in primary external granule layer cells showed that netrin-1 differentially increased MT dynamics in the GC with more MT growth in the distal than the proximal region of the GC during repulsion, and knockdown of either UNC5C or TUBB3 abolished the netrin-1 effect. Together, these data indicate that the disengagement of UNC5C with polymerized TUBB3 plays an essential role in netrin-1/UNC5C-mediated axon repulsion.SIGNIFICANCE STATEMENT Proper regulation of microtubule (MT) dynamics in the growth cone plays an important role in axon guidance. However, whether guidance cues modulate MT dynamics directly or indirectly is unclear. Here, we report that dissociation of UNC5C and polymerized TUBB3, the highly dynamic β-tubulin isoform in neurons, is essential for netrin-1/UNC5C-promoted axon repulsion. These results not only provide a working model of direct modulation of MTs by guidance cues in growth cone navigation but also help us to understand molecular mechanisms underlying developmental brain disorders associated with TUBB3 mutations.

Dataset Information

Pioneer midbrain longitudinal axons navigate using a balance of Netrin attraction and Slit repulsion.

Background

Results

Conclusions

Publications

Pioneer midbrain longitudinal axons navigate using a balance of Netrin attraction and Slit repulsion.

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