Identification of domains of netrin UNC-6 that mediate attractive and repulsive guidance and responses from cells and growth cones - PubMed (original) (raw)

Comparative Study

Identification of domains of netrin UNC-6 that mediate attractive and repulsive guidance and responses from cells and growth cones

Yoo-shick Lim et al. J Neurosci. 2002.

Abstract

Netrin UNC-6 is a protein secreted from ventral cells that guides cell and growth cone migrations in Caenorhabditis elegans. Previously it was shown that UNC-6 domain V-2 regulates dorsal guidance activity and domain C regulates an activity that prevents the branching of axons when they respond to the N-terminal domains. Because these results indicate that the biological activities of UNC-6 are mediated through specific domains, we systematically examined each UNC-6 domain for guidance activities. Transgenic animals expressing UNC-6 derivatives with domain deletions and mutants with selective unc-6 loss-of-function mutations were analyzed. The results indicate that the VI, V-2, and V-3 domains are primarily required for dorsal migrations and the VI and V-3 domains are required for ventral migrations. These domains are likely important for responses mediated by the UNC-5 and UNC-40 receptors, respectively. Deletion of V-3 and a V-3 point mutation selectively affect either cell or growth cone migrations, indicating that each migration requires unique interactions with UNC-6. Deletion of domain VI or of a conserved eight amino acid motif within VI causes loss of all UNC-6 guidance activities, and mutations within domain VI selectively affect different guidance activities, suggesting that domain VI regulates each response to UNC-6. We propose that individual UNC-6 domains mediate different signals, which act in parallel to regulate the morphological changes necessary for guidance.

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Figures

Fig. 1.

Fig. 1.

Schematic representation of the netrin UNC-6 protein and UNC-6 derivatives used in this study. UNC-6 comprises an N-terminal domain VI (residues 1–268) similar to the N-terminal domain VI of laminin subunits, three cysteine-rich repeats (residues 269–437) similar to those of domain V of laminin subunits, and a C-terminal domain named C (residues 438–591) that is not found in laminins but is phylogenetically conserved among other extracellular proteins. An epitope tag comprising three tandem copies of the HA epitope was engineered into a site immediately after the predicted signal peptide (SP).

Fig. 2.

Fig. 2.

Summary of cell and axon positions.A, Cell bodies and axons of representative sensory and motor neurons. The migration of the axons of DA and DB motor neurons and of SDQR were assayed to measure the ability of _unc-6_mutations and transgenes to guide dorsal axon migrations. Only DA motor neurons are represented for simplicity. PVCL and AVM axons were assayed to measure ventral axon migrations. B, Migrating mesodermal cells, the distal tip cells, and the anchor cell were assayed to measure dorsal and ventral cell migrations, respectively. Details of the phenotypes of these cells in unc-5, unc-6, and unc-40 mutants were reported byHedgecock et al. (1990). Anterior is to the left; dorsal is at top.

Fig. 3.

Fig. 3.

Dorsal axon migrations of DA and DB motor neurons.A, In wild-type animals, the DA and DB cell bodies (arrowheads) are positioned along the ventral nerve cord, and each has an axon that migrates longitudinally along the ventral nerve cord (vc) and an axon that migrates circumferentially to the dorsal nerve cord (dc). B, In some mutants, the dorsal circumferential migration defects are relatively mild. In this_unc-40_ mutant, a single axon (arrows) has abnormally migrated at the dorsal sublateral position and fails to reach the dorsal nerve cord. C, In unc-6_mutants, the axons rarely reach the dorsal nerve cord. In this_unc-6 mutant, most axons turn and migrate at the ventral sublateral and lateral positions (arrows). Anterior is to the left; dorsal is at top. Scale bars, 25 μm.

Fig. 4.

Fig. 4.

Ventral axon migrations of PVC in the tail region.A, In wild-type animals, the PVC neurons extend an axon ventrally along the lumbar commissure to the ventral nerve cord (vc). The left PVC neuron (PVCL) is shown. B, In unc-6 mutants, the PVC axons often migrate at the lateral position instead of directly entering the ventral nerve cord. In this animal, the axon from the left PVC neuron (PVCL) has migrated laterally, whereas the axon from the right PVC neuron (PVCR) has correctly migrated to the ventral nerve cord. Anterior is to the left; dorsal is at top. Scale bars, 10 μm.

Fig. 5.

Fig. 5.

A summary of the proposed function for each of the UNC-6 domains. The combination of domains elicits parallel signals that together mediate the different cytoskeletal changes necessary for guidance.

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