A perspective on SOAL, a stochastic model of neuronal outgrowth.

A functional nervous system requires neuronal connections to be made in a highly detailed and stereotypic manner. During development, neurons extend processes that can branch, travel in different directions, and form elaborate patterns. These patterns are essential for forming proper connections. Patterns of outgrowth are produced by complex molecular events that cause a fluid membrane to move. The collective impact of dynamic fluctuating events at the microscale cause the patterns of outgrowth observed at the macroscale. Patterning is genetically controlled, but the effects genes have on membrane movement and patterning are not well understood. To better understand how genes control outgrowth patterns, I propose a statistically-oriented asymmetric localization (SOAL) model. This model is based on the theory that receptor-mediated outgrowth activity is stochastically oriented and when the system is at equilibrium there is an equal probability of outgrowth being oriented in any direction. This concept allows a statistical mechanics approach that can correlate the microscale events of outgrowth to the observed macroscale patterns. Proof-of-concept experiments suggest this approach can be used to study the effect genes have on outgrowth patterns. The SOAL model also provides a new theoretical framework for conceptualizing guidance. According to the model, outgrowth activity becomes asymmetrically localized to the neuron's surface in a statistically dependent manner. Extracellular cues regulate the probability of outgrowth along the surface and the orientation of outgrowth fluctuates across the surface over time. This creates a directional bias that allows the growth cone to navigate in reference to the composition of extracellular cues.

CED-10/Rac1 mediates axon guidance by regulating the asymmetric distribution of MIG-10/lamellipodin.

Axon migrations are guided by extracellular cues that induce asymmetric outgrowth activity in the growth cone. Several intracellular signaling proteins have been implicated in the guidance response. However, how these proteins interact to generate asymmetric outgrowth activity is unknown. Here, we present evidence that in C. elegans, the CED-10/Rac1 GTPase binds to and causes asymmetric localization of MIG-10/lamellipodin, a protein that regulates actin polymerization and has outgrowth-promoting activity in neurons. Genetic analysis indicates that mig-10 and ced-10 function together to orient axon outgrowth. The RAPH domain of MIG-10 binds to activated CED-10/Rac1, and ced-10 function is required for the asymmetric MIG-10 localization that occurs in response to the UNC-6/netrin guidance cue. We also show that asymmetric localization of MIG-10 in growth cones is associated with asymmetric concentrations of f-actin and microtubules. These results suggest that CED-10/Rac1 is asymmetrically activated in response to the UNC-6/netrin signal and thereby causes asymmetric recruitment of MIG-10/lamellipodin. We propose that the interaction between activated CED-10/Rac1 and MIG-10/lamellipodin triggers local cytoskeletal assembly and polarizes outgrowth activity in response to UNC-6/netrin.

Assembly and tissue functions of early embryonic laminins and netrins.

Vertebrate laminins and netrins share N-terminal domain structure, but appear to be only distantly related. Both families can be divided into different subfamilies on the basis of structural considerations. Recent observations suggest that specific laminin and netrin members have developmental functions that are highly conserved across species. Vertebrate laminin-1 (alpha1beta1gamma1) and laminin-10 (alpha5beta1gamma1), like the two Caenorhabditis elegans laminins, are embryonically expressed and are essential for basement membrane assembly. Basement membrane assembly is a cooperative process in which laminins polymerize through their LN domains and anchor to the cell surface through their G domains; this leads to cell signaling through integrins and dystroglycan (and possibly other receptors) recruited to the adherent laminin. Netrins may associate with this network through heterotypic LN domain interactions. Vertebrate netrin-1, like invertebrate UNC-6/netrins, is well known as an extracellular guidance cue that directs axon migration towards or away from the ventral midline. It also regulates cell adhesions and migrations, probably as a basement membrane component. Although sharing structural features, these two vertebrate protein families are quite distinct, having both retained members that mediate the ancestral developmental functions.

Neurodevelopment: UNC-40/DCC and the Patterning of Neural Circuits.

A new study in Caenorhabditis elegans suggests the ubiquitin-proteasome system promotes degradation of the netrin receptor UNC-40 in a particular neuron only in one sex, leading to sex-specific patterns of synaptic connections.

RPM-1, a Caenorhabditis elegans protein that functions in presynaptic differentiation, negatively regulates axon outgrowth by controlling SAX-3/robo and UNC-5/UNC5 activity.

Changes in axon outgrowth patterns are often associated with synaptogenesis. Members of the conserved Pam/Highwire/RPM-1 protein family have essential functions in presynaptic differentiation. Here, we show that Caenorhabditis elegans RPM-1 negatively regulates axon outgrowth mediated by the guidance receptors SAX-3/robo and UNC-5/UNC5. Loss-of-function rpm-1 mutations cause a failure to terminate axon outgrowth, resulting in an overextension of the longitudinal PLM axon. We observe that PLM overextension in rpm-1 mutants is suppressed by sax-3 and unc-5 loss-of-function mutations. PLM axon overextension is also induced by SAX-3 overexpression, and the length of extension is enhanced by loss of rpm-1 function or suppressed by loss of unc-5 function. We also observe that loss of rpm-1 function in genetic backgrounds sensitized for guidance defects disrupts ventral AVM axon guidance in a SAX-3-dependent manner and enhances dorsal guidance of DA and DB motor axons in an UNC-5-dependent manner. Loss of rpm-1 function alters expression of the green fluorescent protein (GFP)-tagged proteins, SAX-3::GFP and UNC-5::GFP. RPM-1 is known to regulate axon termination through two parallel genetic pathways; one involves the Rab GEF (guanine nucleotide exchange factor) GLO-4, which regulates vesicular trafficking, and another that involves the F-box protein FSN-1, which mediates RPM-1 ubiquitin ligase activity. We show that glo-4 but not fsn-1 mutations affect axon guidance in a manner similar to loss of rpm-1 function. Together, the results suggest that RPM-1 regulates axon outgrowth affecting axon guidance and termination by controlling the trafficking of the UNC-5 and SAX-3 receptors to cell membranes.

CLEC-38, a transmembrane protein with C-type lectin-like domains, negatively regulates UNC-40-mediated axon outgrowth and promotes presynaptic development in Caenorhabditis elegans.

In the developing nervous system, axons respond to various guidance cues to find their targets. The effects guidance cues have on an axon may change as an axon undergoes morphological changes, such as branching, turning, and synapse formation. The means by which these changes are regulated are not well understood. In Caenorhabditis elegans, the UNC-40/DCC (deleted in colorectal cancer) receptor mediates responses to the UNC-6/netrin guidance cue. Here, we show that CLEC-38, a protein with predicted transmembrane and C-type lectin-like domains, regulates UNC-40-mediated axon outgrowth as well as the organization of presynaptic terminals. We observe that, in genetic backgrounds sensitized for axon guidance defects, loss of clec-38 function can suppress defects in an UNC-40-dependent manner. Within migrating axons, clec-38 acts cell autonomously. Furthermore, loss of clec-38 function alters UNC-40::GFP (green fluorescent protein) expression. We also observe that loss of clec-38 function disrupts presynaptic patterning in animals with normal axon guidance and that there are genetic interactions between clec-38 and rpm-1, which encodes a protein implicated in regulating presynaptic assembly and axon morphology. We suggest CLEC-38 plays a role in promoting synapse assembly and refining axon outgrowth activity.

Axon guidance: ephrins at WRK on the midline.

Recent findings indicate that the embryonic motor neurons act as gatekeepers to regulate midline crossing during development of the nematode Caenorhabditis elegans. The newly identified protein WRK-1 and ephrins cooperate to prevent longitudinal axons from crossing the midline.

UNC-6/netrin and SLT-1/slit guidance cues orient axon outgrowth mediated by MIG-10/RIAM/lamellipodin.

BACKGROUND: Axon migrations are guided by extracellular cues that can act as repellants or attractants. However, the logic underlying the manner through which attractive and repulsive responses are determined is unclear. Many extracellular guidance cues, and the cellular components that mediate their signals, have been implicated in both types of responses. RESULTS: Genetic analyses indicate that MIG-10/RIAM/lamellipodin, a cytoplasmic adaptor protein, functions downstream of the attractive guidance cue UNC-6/netrin and the repulsive guidance cue SLT-1/slit to direct the ventral migration of the AVM and PVM axons in C. elegans. Furthermore, overexpression of MIG-10 in the absence of UNC-6 and SLT-1 induces a multipolar phenotype with undirected outgrowths. Addition of either UNC-6 or SLT-1 causes the neurons to become monopolar. Moreover, the ability of UNC-6 or SLT-1 to direct the axon ventrally is enhanced by the MIG-10 overexpression. We also demonstrate that an interaction between MIG-10 and UNC-34, a protein that promotes actin-filament extension, is important in the response to guidance cues and that MIG-10 colocalizes with actin in cultured cells, where it can induce the formation of lamellipodia. CONCLUSIONS: We conclude that MIG-10 mediates the guidance of AVM and PVM axons in response to the extracellular UNC-6 and SLT-1 guidance cues. The attractive and repulsive guidance cues orient MIG-10-dependant axon outgrowth to cause a directional response.

Axon pruning: C. elegans makes the cut.

Axon pruning has recently been described in the simple nervous system of the nematode Caenorhabditis elegans. Generating excess processes and pruning may be a phylogenetically conserved feature reflecting a flexibility to modify neural circuits.

A Statistically-Oriented Asymmetric Localization (SOAL) Model for Neuronal Outgrowth Patterning by Caenorhabditis elegans UNC-5 (UNC5) and UNC-40 (DCC) Netrin Receptors.

Neurons extend processes that vary in number, length, and direction of "outgrowth". Extracellular cues help determine outgrowth patterns. In Caenorhabditis elegans, neurons respond to the extracellular UNC-6 (netrin) cue via UNC-40 (DCC) and UNC-5 (UNC5) receptors. Previously, we presented evidence that UNC-40 asymmetric localization at the plasma membrane is self-organizing, and that UNC-40 can localize and mediate outgrowth at randomly selected sites. Here, we provide further evidence for a statistically-oriented asymmetric localization (SOAL) model in which UNC-5 receptor activity affects patterns of axon outgrowth by regulating UNC-40 asymmetric localization. According to the SOAL model, the direction of outgrowth activity fluctuates across the membrane over time. Random walk modeling predicts that increasing the degree to which the direction of outgrowth fluctuates will decrease the outward displacement of the membrane. By differentially affecting the degree to which the direction of outgrowth activity fluctuates over time, extracellular cues can produce different rates of outgrowth along the surface and create patterns of "extension". Consistent with the SOAL model, we show that unc-5 mutations alter UNC-40 asymmetric localization, increase the degree to which the direction of outgrowth fluctuates, and reduce the extent of outgrowth in multiple directions relative to the source of UNC-6 These results are inconsistent with current models, which predict that UNC-5 mediates a "repulsive" response to UNC-6 Genetic interactions suggest that UNC-5 acts through the UNC-53 (NAV2) cytoplasmic protein to regulate UNC-40 asymmetric localization in response to both the UNC-6 and EGL-20 (Wnt) extracellular cues.

Moving around in a worm: netrin UNC-6 and circumferential axon guidance in C. elegans.

How does an extracellular guidance molecule direct multiple growth cones to different positions? The answer is important for understanding the development of complex neural connections. UNC-6 is a member of the netrin family of guidance proteins. It has phylogenetically conserved domains that mediate its different guidance and branching activities. In the Caenorhabditis elegans embryo, UNC-6 is secreted ventrally and a pattern of circumferential axon tracts develops as pioneer growth cones bearing UNC-5 and UNC-40 receptors are directed towards, or away from, the ventral sources. Following the first migrations, UNC-6 from additional sources allows more complex migration patterns to emerge. In addition, at specific dorsoventral positions, locally restricted extracellular molecules alter growth cone responses to UNC-6, causing circumferentially migrating growth cones to turn and longitudinal nerves to develop. These observations show that extracellular guidance molecules can direct complex arrangements of migrating growth cones in vivo by eliciting different types of responses, by spatially and temporally regulating their expression and by working in concert with other extracellular molecules.

The Genetics of Axon Guidance and Axon Regeneration in Caenorhabditis elegans.

The correct wiring of neuronal circuits depends on outgrowth and guidance of neuronal processes during development. In the past two decades, great progress has been made in understanding the molecular basis of axon outgrowth and guidance. Genetic analysis in Caenorhabditis elegans has played a key role in elucidating conserved pathways regulating axon guidance, including Netrin signaling, the slit Slit/Robo pathway, Wnt signaling, and others. Axon guidance factors were first identified by screens for mutations affecting animal behavior, and by direct visual screens for axon guidance defects. Genetic analysis of these pathways has revealed the complex and combinatorial nature of guidance cues, and has delineated how cues guide growth cones via receptor activity and cytoskeletal rearrangement. Several axon guidance pathways also affect directed migrations of non-neuronal cells in C. elegans, with implications for normal and pathological cell migrations in situations such as tumor metastasis. The small number of neurons and highly stereotyped axonal architecture of the C. elegans nervous system allow analysis of axon guidance at the level of single identified axons, and permit in vivo tests of prevailing models of axon guidance. C. elegans axons also have a robust capacity to undergo regenerative regrowth after precise laser injury (axotomy). Although such axon regrowth shares some similarities with developmental axon outgrowth, screens for regrowth mutants have revealed regeneration-specific pathways and factors that were not identified in developmental screens. Several areas remain poorly understood, including how major axon tracts are formed in the embryo, and the function of axon regeneration in the natural environment.

The C domain of netrin UNC-6 silences calcium/calmodulin-dependent protein kinase- and diacylglycerol-dependent axon branching in Caenorhabditis elegans.

Second messenger systems mediate neuronal responses to extracellular factors that elicit axon branching, turning, and guidance. We found that mutations in Caenorhabditis elegans that affect components of second messenger systems, a G-protein subunit, phospholipase Cbeta, diacylglycerol (DAG) kinase, and calcium/calmodulin-dependent protein kinase (CaMKII), have no obvious effect on axon responses to UNC-6 except in animals in which the N-terminal fragment, UNC-6DeltaC, is expressed. In these animals, the mutations enhance or suppress ectopic branching of certain axons. Netrin UNC-6 is an extracellular protein that guides circumferential migrations, and UNC-6DeltaC has UNC-6 guidance activity. We propose that the guidance response elicited by the UNC-6 N-terminal domains involves mechanisms that can induce branching that is sensitive to CaMKII- and DAG-dependent signaling, and that the UNC-6 C domain is required in cis to the N-terminal domains to silence the branching and to maintain proper axon morphology.

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

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.

SAX-3 (Robo) and UNC-40 (DCC) regulate a directional bias for axon guidance in response to multiple extracellular cues.

Axons in Caenorhabditis elegans are guided by multiple extracellular cues, including UNC-6 (netrin), EGL-20 (wnt), UNC-52 (perlecan), and SLT-1 (slit). How multiple extracellular cues determine the direction of axon guidance is not well understood. We have proposed that an axon's response to guidance cues can be modeled as a random walk, i.e., a succession of randomly directed movement. Guidance cues dictate the probability of axon outgrowth activity occurring in each direction, which over time creates a directional bias. Here we provide further evidence for this model. We describe the effects that the UNC-40 (DCC) and SAX-3 (Robo) receptors and the UNC-6, EGL-20, UNC-52, and SLT-1 extracellular cues have on the directional bias of the axon outgrowth activity for the HSN and AVM neurons. We find that the directional bias created by the cues depend on UNC-40 or SAX-3. UNC-6 and EGL-20 affect the directional bias for both neurons, whereas UNC-52 and SLT-1 only affect the directional bias for HSN and AVM, respectively. The direction of the bias created by the loss of a cue can vary and the direction depends on the other cues. The random walk model predicts this combinatorial regulation. In a random walk a probability is assigned for each direction of outgrowth, thus creating a probability distribution. The probability distribution for each neuron is determined by the collective effect of all the cues. Since the sum of the probabilities must equal one, each cue affects the probability of outgrowth in multiple directions.

Extracellular matrix regulates UNC-6 (netrin) axon guidance by controlling the direction of intracellular UNC-40 (DCC) outgrowth activity.

How extracellular molecules influence the direction of axon guidance is poorly understood. The HSN axon of Caenorhabditis elegans is guided towards a ventral source of secreted UNC-6 (netrin). The axon's outgrowth response to UNC-6 is mediated by the UNC-40 (DCC) receptor. We have proposed that in response to the UNC-6 molecule the direction of UNC-40-mediated axon outgrowth is stochastically determined. The direction of guidance is controlled by asymmetric cues, including the gradient of UNC-6, that regulate the probability that UNC-40-mediated axon outgrowth is directed on average, over time, in a specific direction. Here we provide genetic evidence that a specialized extracellular matrix, which lies ventral to the HSN cell body, regulates the probability that UNC-40-mediated axon outgrowth will be directed ventrally towards the matrix. We show that mutations that disrupt the function of proteins associated with this matrix, UNC-52 (perlecan), UNC-112 (kindlin), VAB-19 (Kank), and UNC-97 (PINCH), decrease the probability of UNC-40-mediated axon outgrowth in the ventral direction, while increasing the probability of outgrowth in the anterior and posterior directions. Other results suggest that INA-1 (α integrin) and MIG-15 (NIK kinase) signaling mediate the response in HSN. Although the AVM axon also migrates through this matrix, the mutations have little effect on the direction of AVM axon outgrowth, indicating that responses to the matrix are cell-specific. Together, these results suggest that an extracellular matrix can regulate the direction of UNC-6 guidance by increasing the probability that UNC-40-mediated axon outgrowth activity will be oriented in a specific direction.

Experimental evidence for UNC-6 (netrin) axon guidance by stochastic fluctuations of intracellular UNC-40 (DCC) outgrowth activity.

How the direction of axon guidance is determined is not understood. In Caenorhabditis elegans the UNC-40 (DCC) receptor mediates a response to the UNC-6 (netrin) guidance cue that directs HSN axon development. UNC-40 becomes asymmetrically localized within the HSN neuron to the site of axon outgrowth. Here we provide experimental evidence that the direction of guidance can be explained by the stochastic fluctuations of UNC-40 asymmetric outgrowth activity. We find that the UNC-5 (UNC5) receptor and the cytoskeletal binding protein UNC-53 (NAV2) regulate the induction of UNC-40 localization by UNC-6. If UNC-40 localization is induced without UNC-6 by using an unc-53 mutation, the direction of UNC-40 localization undergoes random fluctuations. Random walk models describe the path made by a succession of randomly directed movement. This model was experimentally tested using mutations that affect Wnt/PCP signaling. These mutations inhibit UNC-40 localization in the anterior and posterior directions. As the axon forms in Wnt/PCP mutants, the direction of UNC-40 localization randomly fluctuates; it can localize in either the anterior, posterior, or ventral direction. Consistent with a biased random walk, over time the axon will develop ventrally in response to UNC-6, even though at a discrete time UNC-40 localization and outgrowth can be observed anterior or posterior. Also, axon formation is slower in the mutants than in wild-type animals. This is also consistent with a random walk since this model predicts that the mean square displacement (msd) will increase only linearly with time, whereas the msd increases quadratically with time for straight-line motion.

Axon response to guidance cues is stimulated by acetylcholine in Caenorhabditis elegans.

Gradients of acetylcholine can stimulate growth cone turning when applied to neurons grown in culture, and it has been suggested that acetylcholine could act as a guidance cue. However, the role acetylcholine plays in directing axon migrations in vivo is not clear. Here, we show that acetylcholine positively regulates signaling pathways that mediate axon responses to guidance cues in Caenorhabditis elegans. Mutations that disrupt acetylcholine synthesis, transportation, and secretion affect circumferential axon guidance of the AVM neuron and in these mutants exogenously supplied acetylcholine improves AVM circumferential axon guidance. These effects are not observed for the circumferential guidance of the DD and VD motor neuron axons, which are neighbors of the AVM axon. Circumferential guidance is directed by the UNC-6 (netrin) and SLT-1 (slit) extracellular cues, and exogenously supplied acetylcholine can improve AVM axon guidance in mutants when either UNC-6- or SLT-1-induced signaling is disrupted, but not when both signaling pathways are perturbed. Not in any of the mutants does exogenously supplied acetylcholine improve DD and VD axon guidance. The ability of acetylcholine to enhance AVM axon guidance only in the presence of either UNC-6 or SLT-1 indicates that acetylcholine potentiates UNC-6 and SLT-1 guidance activity, rather than acting itself as a guidance cue. Together, our results show that for specific neurons acetylcholine plays an important role in vivo as a modulator of axon responses to guidance cues.