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Recent studies in vertebrates and Caenorhabditis elegans have reshaped models of how the axon guidance cue UNC-6/Netrin functions in dorsal-ventral axon guidance, which was traditionally thought to form a ventral-to-dorsal concentration gradient that was actively sensed by growing axons. In the vertebrate spinal cord, floorplate Netrin1 was shown to be largely dispensable for ventral commissural growth. Rather, short range interactions with Netrin1 on the ventricular zone radial glial stem cells was shown to guide ventral commissural axon growth. In C. elegans, analysis of dorsally-migrating growth cones during outgrowth has shown that growth cone polarity of filopodial extension is separable from the extent of growth cone protrusion. Growth cones are first polarized by UNC-6/Netrin, and subsequent regulation of protrusion by UNC-6/Netrin is based on this earlier-established polarity (the Polarity/Protrusion model). In both cases, short-range or even haptotactic mechanisms are invoked: in vertebrate spinal cord, interactions of growth cones with radial glia expressing Netrin-1; and in C. elegans, a potential close-range interaction that polarizes the growth cone. To explore potential short-range and long-range functions of UNC-6/Netrin, a potentially membrane-anchored transmembrane UNC-6 (UNC-6(TM)) was generated by genome editing. unc-6(tm) was hypomorphic for dorsal VD/DD axon pathfinding, indicating that it retained some unc-6 function. Polarity of VD growth cone filopodial protrusion was initially established in unc-6(tm), but was lost as the growth cones migrated away from the unc-6(tm) source in the ventral nerve cord. In contrast, ventral guidance of the AVM and PVM axons was equally severe in unc-6(tm) and unc-6(null). Together, these results suggest that unc-6(tm) retains short-range functions but lacks long-range functions due to reduced secreted UNC-6. Ectopic unc-6(+) expression from non-ventral sources did not dramatically perturb dorsal VD growth cone polarity or axon outgrowth, suggesting that ectopic UNC-6 cannot redirect polarity once it is established in the VD/DD neurons. This is not what would be expected of a growth cone dynamically reading a gradient of UNC-6, but is consistent with the Polarity/protrusion model of growth cone guidance away from UNC-6/Netrin. Author summary: In the developing nervous system, neurons extend thin cellular processes called axons that make precise contacts with other neurons in elaborate networks. The structure of these axon networks underlies the function of the nervous system. Thus precise axon guidance is critical during development to produce a functional nervous system. Axons are guided by the growth cone, a dynamic structure at the distal tup of the axon that senses and responds to signals in the environment that instruct axon guidance. One such signal if UNC-6/Netrin, a conserved, laminin-like secreted molecule that guides many aspects of axon guidance. In the nematode Caenorhabditis elegans, UNC-6 controls dorsal-ventral axon guidance of motor neurons and sensory neurons. The canonical textbook model of UNC-6/Netrin function involved the formation of a gradient of secreted UNC-6/Netrin in the ventral-to-dorsal axis which growth cones dynamically sensed and responded by growing up or down the gradient. The gradient model does not explain recent studies in vertebrates and C. elegans that have shown close-range or contact-mediated events that more resemble cell polarity events than dynamic reading of a gradient. In C. elegans, the growth cone of the VD axon is polarized dorsally by UNC-6/Netrin. UNC-6/Netrin then maintains this polarity and regulates protrusion based upon this polarity, stimulating protrusion dorsally and inhibiting protrusion ventrally, resulting in net dorsal outgrowth. This Polarity/Protrusion model resembles events that take place in ventral growth of sensory axons, the Statistically-Oriented Asymmetric Localization (SOAL) model. The Polarity/Protrusion model predicts that a short-range UNC-6 signal polarizes the VD growth cone, and that secreted, diffusible UNC-6/Netrin maintains initial polarity and regulates protrusion. This model is critically tested here by the construction of a membrane-anchored UNC-6 genome edit predicted to not produce any diffusible UNC-6, thus blocking any long-range function. Data here show that VD growth cones are initially polarized corerectly in unc-6(tm), but that this polarity is lost as the growth cones move dorsally away from the UNC-6(TM) source. This is consistent with a short-range interaction that polarizes the growth cone, and a long-range function needed to maintain polarity. Furthermore, ectopic expression of diffusible UNC-6 from ectopic sources did not perturb VD growth cone polarity, suggesting that the initial polarity event cannot be altered by later diffusible UNC-6/Netrin, as would be expected with a gradient model. These results are consistent with the Polarity/Protrusion model of UNC-6 function. [ABSTRACT FROM AUTHOR]

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In the developing nervous system, axons are guided to their targets by the growth cone. Lamellipodial and filopodial protrusions from the growth cone underlie motility and guidance. Many molecules that control lamellipodia and filopodia formation, actin organization, and axon guidance have been identified, but it remains unclear how these molecules act together to control these events. Experiments are described here that indicate that, in Caenorhabditis elegans, two WH2-domain-containing activators of the Arp2/3 complex, WVE-1/WAVE and WSP-1/WASP, act redundantly in axon guidance and that GEX-2/Sra-1 and GEX-3/ Kette, molecules that control WAVE activity, might act in both pathways. WAVE activity is controlled by Rac GTPases, and data are presented here that suggest WVE-1/WAVE and CED-10/Rac act in parallel to a pathway containing WSP-1/WASP and MIG-2/RhoG. Furthermore, results here show that the CED-10/ WVE-1 and MIG-2/WSP-1 pathways act in parallel to two other molecules known to control lamellipodia and filopodia and actin organization, UNC-115/abLIM and UNC-34/Enabled. These results indicate that at least three actin-modulating pathways act in parallel to control actin dynamics and lamellipodia and filopodia formation during axon guidance (WASP--WAVE, UNC-115/abLIM, and UNC-34/Enabled).

Rac GTPases are key regulators of cell shape and cytoskeletal organization. While some regulators of Rac activity are known, such as GTPase-activating proteins (GAPs) that repress Rac activity, other Rac regulators remain to be identified. The novel Caenorhabditis elegans WD-repeat protein SWAN-1 was identified in a yeast two-hybrid screen with the LIM domains of the Rac effector UNC-115/abLIM. SWAN-1 was found to also associate physically with Rac GTPases. The swan-1(ok267) loss-of-function mutation suppressed defects caused by the hypomorphic ced-10(n1993) allele and enhanced ectopic lamellipodia and filopodia formation induced by constitutively active Rac in C. elegans neurons. Furthermore, SWAN-1 (+) transgenic expression suppressed the effects of overactive Rac, including ectopic lamellipodia and filopodia formation in C. elegans neurons, ectopic lamellipodia formation in cultured mammalian fibroblasts, and cell polarity and actin cytoskeleton defects in yeast. These studies indicate that SWAN-1 is an inhibitor of Rac GTPase function in cellular morphogenesis and cytoskeletal organization. While broadly conserved across species, SWAN-1 family members show no sequence similarity to previously known Rac inhibitors.

Many genes that affect axon pathfinding and cell migration have been identified. Mechanisms by which these genes and the molecules they encode interact with one another in pathways and networks to control developmental events are unclear. Rac GTPases, the cytoskeletal signaling molecule Enabled, and NIK kinase have all been implicated in regulating axon pathfinding and cell migration. Here we present evidence that, in Caenorhabditis elegans, three Rac GTPases, CED-10, RAC-2, and MIG-2, define three redundant pathways that each control axon pathfinding, and that the NIK kinase MIG-15 acts in each Rac pathway. Furthermore, we show that the Enabled molecule UNC-34 defines a fourth partially redundant pathway that acts in parallel to Rac/MIG-15 signaling in axon path finding. Enabled and the three Racs also act redundantly to mediate AQR and PQR neuronal cell migration. The Racs and UNC-34 Ena might all control the formation of actin-based protrusive structures (lamellipodia and filopodia) that mediate growth cone outgrowth and cell migration. MIG-15 does not act with the three Racs in execution of cell migration. Rather, MIG-15 affects direction of PQR neuronal migration, similar to UNC-40 and DPY-19, which control initial Q cell polarity, and Wnt signaling, which acts later to control Q cell-directed migration. MIG-2 Rac, which acts with CED-10 Rac, RAC-2 Rac, and UNC-34 Ena in axon pathfinding and cell migration, also acts with MIG-15 in PQR directional migration.

Mutations in the unc-39 gene of C. elegans lead to migration and differentiation defects in a subset of mesodermal and ectodermal cells, including muscles and neurons. Defects include mesodermal specification and differentiation as well a neuronal migration and axon pathfinding defects. Molecular analysis revealed that unc-39 corresponds to the previously named gene ceh-35 and that the UNC-39 protein belongs to the Six4/5 family of homeodomain transcription factors and is similar to human Six5, a protein implicated in the pathogenesis of type I myotonic dystrophy (DM1). We show that human Six5 and UNC-39 are functional homologs, suggesting that further characterization of the C. elegans unc-39 gene might provide insight into the etiology of DM1. Keywords: C. elegans; unc-39; Myotonic dystrophy

Mutation in the Caenorhabditis elegans gene osm-6 was previously shown to result in defects in the ultrastructure of sensory cilia and defects in chemosensory and mechanosensory behaviors. We have cloned osm-6 by transposon tagging and transformation rescue and have identified molecular lesions associated with five osm-6 mutations. The osm-6 gene encodes a protein that is 40% identical in amino acid sequence to a predicted mammalian protein of unknown function. We fused osm-6 with the gene for green fluorescent protein (GFP); the fusion gene rescued the osm-6 mutant phenotype and showed accumulation of GFP in ciliated sensory neurons exclusively. The OSM-6::GFP protein was localized to cytoplasm, including processes and dendritic endings where sensory cilia are situated. Mutations in other genes known to cause ciliary defects led to changes in the appearance of OSM-6::GFP in dendritic endings or, in the case of daf19, reduced OSM-6::GFP accumulation. We conclude from an analysis of genetic mosaics that osm-6 acts cell autonomously in affecting cilium structure.

Analysis of the role of the Caenorhabditis elegans gene mec-8 in muscle and sensory neuronal activities reveals mutant alleles of mec-8 that cause defects in mechano- and chemosensory functions in C. elegans. Strong mutations in mec-8 cause larval lethality and produce cold-sensitive structural lesions in body muscles. Mutations in the mec-8 phenotype unc-52 cause progressive paralysis and disruption of muscle structure organization. Four suppressors of mec-8 and unc-52 that are encoded by smu-1 and smu-2 genes are identified.

Researchers isolated a new mutation, called Abdominal-B Corset (Abd-B Corset), which is the result of deletion that leaves 15kb of the more removed 3' regulatory sequences exactly next to the gene, but withdraws 45 Kb of the more removed 3' regulatory factors. The study has informed a construct of the 3' regulatory area of Abd-13 founded on reiterated cell type-specific factors determined by position-sensitive stimulating factors next to it.

Abstract Background While many molecules involved in axon guidance have been identified, the cellular and molecular mechanisms by which these molecules regulate growth cone morphology during axon outgrowth remain to be elucidated. The actin cytoskeleton of the growth cone underlies the formation of lamellipodia and filopodia that control growth cone outgrowth and guidance. The role of the Arp2/3 complex in growth cone filopodia formation has been controversial, and other mechanisms of growth cone filopodia formation remain to be described. Results Here we show that mutations in genes encoding the Arp2/3 complex (arx genes) caused defects in axon guidance. Analysis of developing growth cones in vivo showed that arx mutants displayed defects in filopodia and reduced growth cone size. Time-lapse analysis of growth cones in living animals indicated that arx mutants affected the rate of growth cone filopodia formation but not filopodia stability or length. Two other actin modulatory proteins, UNC-115/abLIM and UNC-34/Enabled, that had been shown previously to affect axon guidance had overlapping roles with Arp2/3 in axon guidance and also affected the rate of filopodia initiation but not stability or length. Conclusion Our results indicate that the Arp2/3 complex is required cell-autonomously for axon guidance and growth cone filopodia initiation. Furthermore, they show that two other actin-binding proteins, UNC-115/abLIM and UNC-34/Enabled, also control growth cone filopodia formation, possibly in parallel to Arp2/3. These studies indicate that, in vivo, multiple actin modulatory pathways including the Arp2/3 complex contribute to growth cone filopodia formation during growth cone outgrowth.