The ENU-3 protein family members function in the Wnt pathway parallel to UNC-6/Netrin to promote motor neuron axon outgrowth in C. elegans.

The axons of the DA and DB classes of motor neurons fail to reach the dorsal cord in the absence of the guidance cue UNC-6/Netrin or its receptor UNC-5 in C. elegans. However, the axonal processes usually exit their cell bodies in the ventral cord in the absence of both molecules. Strains lacking functional versions of UNC-6 or UNC-5 have a low level of DA and DB motor neuron axon outgrowth defects. We found that mutations in the genes for all six of the ENU-3 proteins function to enhance the outgrowth defects of the DA and DB axons in strains lacking either UNC-6 or UNC-5. A mutation in the gene for the MIG-14/Wntless protein also enhances defects in a strain lacking either UNC-5 or UNC-6, suggesting that the ENU-3 and Wnt pathways function parallel to the Netrin pathway in directing motor neuron axon outgrowth. Our evidence suggests that the ENU-3 proteins are novel members of the Wnt pathway in nematodes. Five of the six members of the ENU-3 family are predicted to be single-pass trans-membrane proteins. The expression pattern of ENU-3.1 was consistent with plasma membrane localization. One family member, ENU-3.6, lacks the predicted signal peptide and the membrane-spanning domain. In HeLa cells ENU-3.6 had a cytoplasmic localization and caused actin dependent processes to appear. We conclude that the ENU-3 family proteins function in a pathway parallel to the UNC-6/Netrin pathway for motor neuron axon outgrowth, most likely in the Wnt pathway.

ENU-3 functions in an UNC-6/netrin dependent pathway parallel to UNC-40/DCC/frazzled for outgrowth and guidance of the touch receptor neurons in C. elegans.

BACKGROUND: UNC-6 and SLT-1 guide the migrations of the ventrally directed processes of the AVM and PVM touch receptor neurons and UNC-6 guides the axons of the DA and DB classes of motor neurons in C. elegans. The UNC-6 receptors are UNC-5 and UNC-40. The axon outgrowth defects of a subset of the DB motor neurons in the absence of UNC-5 are enhanced by mutations in enu-3. RESULTS: An enu-3 mutation enhances defects in ventral guidance of the processes of the AVM and PVM touch receptor neurons, the dorsal guidance of the distal tip cell and causes additional architectural defects in axons in unc-40 mutant strains in an UNC-6 dependent manner. These observations suggest that ENU-3 and UNC-40 function in parallel pathways dependent on UNC-6. ENU-3 depends on the presence of UNC-40 for its full effect on motor neuron axon outgrowth. CONCLUSIONS: ENU-3 works in an UNC-6 dependent pathway parallel to UNC-40 in ventral guidance of AVM and PVM and in dorsal guidance of the distal tip cells. Motor neuron axon outgrowth defects are caused by the presence of UNC-40 and the absence of functional UNC-5 or UNC-6 and defects are enhanced by the absence of functional ENU-3.

ENU-3 is a novel motor axon outgrowth and guidance protein in C. elegans.

During the development of the nervous system, the migration of many cells and axons is guided by extracellular molecules. These molecules bind to receptors at the tips of the growth cones of migrating axons and trigger intracellular signaling to steer the axons along the correct trajectories. We have identified a novel mutant, enu-3 (enhancer of Unc), that enhances the motor neuron axon outgrowth defects observed in strains of Caenorhabditis elegans that lack either the UNC-5 receptor or its ligand UNC-6/Netrin. Specifically, the double-mutant strains have enhanced axonal outgrowth defects mainly in DB4, DB5 and DB6 motor neurons. enu-3 single mutants have weak motor neuron axon migration defects. Both outgrowth defects of double mutants and axon migration defects of enu-3 mutants were rescued by expression of the H04D03.1 gene product. ENU-3/H04D03.1 encodes a novel predicted putative trans-membrane protein of 204 amino acids. It is a member of a family of highly homologous proteins of previously unknown function in the C. elegans genome. ENU-3 is expressed in the PVT interneuron and is weakly expressed in many cell bodies along the ventral cord, including those of the DA and DB motor neurons. We conclude that ENU-3 is a novel C. elegans protein that affects both motor axon outgrowth and guidance.

The dual role of the ligand UNC-6/Netrin in both axon guidance and synaptogenesis in C. elegans.

The extracellular cue UNC-6/Netrin is a well-known axon guidance molecule and recently it has also been shown to be involved with localization of pre-synaptic complexes. Working through the UNC-40/DCC/Fra receptor, UNC-6/Netrin promotes the formation of pre-synaptic terminals between the pre-synaptic AIY interneuron and its post-synaptic partner, the RIA interneuron. In the DA9 motor neuron, UNC-6/Netrin has an alternate role promoting the exclusion of pre-synaptic components from the dendrite via its UNC-5-receptor. Surprisingly, the requirement for UNC-5 persists even after DA9 axon migration is complete, because synapses become mis-localized after it is depleted. This observation provides at least a partial explanation for the persistence of UNC-6/Netrin and UNC-5 in the adult nervous system. These activities parallel the previously known bi-functional axon guidance effects of UNC-6/Netrin, since it can attract cells and axons expressing UNC-40/DCC/Fra and repel those expressing UNC-5 alone or in combination with UNC-40. UNC-6/Netrin cooperates with the Wnt family members to exclude synapses from compartments within the DA9 axon, so that they only occur in regions free of the influence of both UNC-6/Netrin and the Wnts. Regulation of both axon guidance and synapse formation by axon guidance cues permits coordination in circuit assembly between pre- and post-synaptic cells.

Netrin, Slit and Wnt receptors allow axons to choose the axis of migration.

One of the challenges to understanding nervous system development has been to establish how a fairly limited number of axon guidance cues can set up the patterning of very complex nervous systems. Studies on organisms with relatively simple nervous systems such as Drosophila melanogaster and C. elegans have provided many insights into axon guidance mechanisms. The axons of many neurons migrate along both the dorsal-ventral (DV) and the anterior-posterior (AP) axes at different phases of development, and in addition they may also cross the midline. Axon migration in the dorsal-ventral (DV) direction is mainly controlled by Netrins with their receptors; UNC-40/DCC and UNC-5, and the Slits with their receptors; Robo/SAX-3. Axon guidance in the anterior-posterior (AP) axis is mainly controlled by Wnts with their receptors; the Frizzleds/Fz. An individual axon may be subjected to opposing attractive and repulsive forces coming from opposite sides in the same axis but there may also be opposing cues in the other axis of migration. All the information from the cues has to be integrated within the growth cone at the leading edge of the migrating axon to elicit a response. Recent studies have provided insight into how this is achieved. Evidence suggests that the axis of axon migration is determined by the manner in which Netrin, Slit and Wnt receptors are polarized (localized) within the neuron prior to axon outgrowth. The same molecules are involved in both axon outgrowth and axon guidance, for at least some neurons in C. elegans, whether the cue is the attractive cue UNC-6/Netrin working though UNC-40/DCC or the repulsive cue SLT-1/Slit working though the receptor SAX-3/Robo (Adler et al., 2006, Chang et al., 2006, Quinn et al., 2006, 2008). The molecules involved in cell signaling in this case are polarized within the cell body of the neuron before process outgrowth and direct the axon outgrowth. Expression of the Netrin receptor UNC-40/DCC or the Slit receptor SAX-3/Robo in axons that normally migrate in the AP direction causes neuronal polarity reversal in a Netrin and Slit independent manner (Levy-Strumpf and Culotti 2007, Watari-Goshima et al., 2007). Localization of the receptors in this case is caused by the kinesin-related VAB-8L which appears to govern the site of axon outgrowth in these neurons by causing receptor localization. Therefore, asymmetric localization of axon guidance receptors is followed by axon outgrowth in vivo using the receptor's normal cue, either attractive, repulsive or unknown cues.