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Molecular Mechanisms Underlying Modulation of UNC-6 and SLT-1 Axon Guidance Signaling in C. elegans

by
Yan Xu
BS, 1997
Nanjing University
Nanjing, P.R. China

Thesis Advisor: William Wadsworth, PhD

Graduate Program: Biochemistry & Molecular Biology

RWJMS Research Tower
Room V-10

Wednesday, August 12, 2009
10:00 am


Abstract

Proper function of the nervous system requires precise connectivity between neurons and their targets which are often separated over long distances. A critical phase to ensure the precision is axon guidance in the early nervous system development. Growth cones located at the leading edge of the migrating axons are guided by the phylogenetically conserved axon guidance cues to reach their appropriate targets. During the growth cone migration, the axon guidance signaling is always subjected to modulation by other signals to achieve accurate growth cone movement. A good example for axon guidance modulation is the midline of the developing central nervous system (CNS). It serves as an important choice point (the intermediate target) for migrating axons and provides information to modulate the growth cone steering. However, the mechanism underlying modulation of the axon guidance signaling is still not fully understood.

In C. elegans, RPM-1, a conserved E3-ubiquitin ligase, was found to negatively regulate the axon guidance receptor UNC-5 and SAX-3 activities and modulate axon outgrowth that affects axon guidance. In order to identify more genes that function with rpm-1 to modulate axon guidance, we performed a suppressor and enhancer screen of axon overextension phenotype caused by loss of function of rpm-1. From this screen, we identified two enhancer genes unc-104 and unc-17. Gene unc-104 encodes a kinesin motor that is required for the antegrade synaptic vesticluar trafficking. Gene unc-17 encodes a vesicular acetylcholine transporter. Loss of both genes causes deficiency in transportation and secretion of the neurotransmitter acetylcholine, which suggests that acetylcholine might play a role in modulating axon guidance signaling.

Indeed, we found that deficiency in synthesis, vesicular transportation, and secretion of acetylcholine causes touch receptor neuron AVM ventral axon guidance defect in C. elegans, which suggests that acetylcholine is involved in promoting AVM axon guidance. The AVM neuron is located at the right lateral side of the C. elegans body wall. At the beginning of the L2 stage, AVM sends its axon ventrally till the ventral nerve cord from where its axon move anteriorly till nerve ring. We found that ventral nerve cord cholinergic motor neurons, the intermediate targets of the AVM axon, secrete acetylcholine for regulating AVM axon guidance. Furthermore, the AVM axon is guided ventrally by two axon guidance signaling pathways, UNC-6/Netrin via its receptor UNC-40/DCC as well as SLT-1/Slit via its receptor SAX-3/robo. We found that acetylcholine acts as a modulator to promote both of UNC-6 and SLT-1 signaling. Moreover, we found that the nicotinic receptors including DEG-3/DES-2 may mediate the function of acetylcholine on AVM axon guidance. Finally, we found that acetylcholine may regulate AVM axon guidance through negative regulation of RPM-1 as well as CLEC-38, a protein with C-type lectin-like domains. Above all, our evidences define that acetylcholine from the intermediate target neurons promotes both UNC-6 and SLT-1 signaling in vivo in C. elegans.


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