Netrin-1: diversity in development

In 1990, the discovery of three Caenorhabditis elegans genes (unc5, unc6, unc40) involved in pioneer axon guidance and cell migration marked a significant advancement in neuroscience research [Hedgecock EM, Culotti JG, Hall DH. The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans. Neuron 1990;4:61-85]. The importance of this molecular guidance system was exemplified in 1994, when the vertebrate orthologue of Unc6, Netrin-1, was discovered to be a key guidance cue for commissural axons projecting toward the ventral midline in the rodent embryonic spinal cord [Serafini T, Kennedy TE, Galko MJ, Mirzayan C, Jessell TM, Tessier-Lavigne M. The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell 1994;78:409-424]. Since then, Netrin-1 has been found to be a critical component of embryonic development with functions in axon guidance, cell migration, morphogenesis and angiogenesis. Netrin-1 also plays a role in the adult brain, suggesting that manipulating netrin signals may have novel therapeutic applications.     

The Caenorhabditis elegans P21-activated kinases are differentially required for UNC-6/netrin-mediated commissural motor axon guidance

P21 activated kinases (PAKs) are major downstream effectors of rac-related small GTPases that regulate various cellular processes. We have identified the new PAK gene max-2 in a screen for mutants disrupted in UNC-6/netrin-mediated commissural axon guidance. There are three Caenorhabditis elegans PAKs. We find that each C. elegans PAK represents a distinct group previously identified in other species. Here we examine their roles in the postembryonic migration of the P cell neuroblasts and the axon guidance of the ventral cord commissural motoneurons (VCCMNs). We find that the two PAKs, max-2 and pak-1, are redundantly required for P cell migration and function with UNC-73/Trio and the rac GTPases (CED-10 and MIG-2). During axon guidance of the VCCMNs, PAK-1 also acts with the rac GTPases, CED-10 and MIG-2, and is completely redundant with MAX-2. Interestingly, we find that unlike MAX-2 activity during P cell migration, for motoneuron axon guidance max-2 is also required in parallel to this PAK-1 pathway, independent of rac GTPase signaling. Finally, we provide evidence that MAX-2 functions downstream of the UNC-6/netrin receptor UNC-5 during axon repulsion and is an integral part of its signaling.     

Navigating the anterior-posterior axis with Wnts

Recent studies have begun to shed light on the molecular guidance cues controlling anterior-posterior axon guidance. Two recent studies in the current issue of Developmental Cell show that Wnts play critical roles in patterning processes and directing neuronal migration in C. elegans. Together with previous findings in vertebrates and flies, these new results establish conserved function of Wnts in A-P guidance.     

Complex network of Wnt signaling regulates neuronal migrations during Caenorhabditis elegans development

Members of the Wnt family of secreted glycoproteins regulate many developmental processes, including cell migration. We and others have previously shown that the Wnts egl-20, cwn-1, and cwn-2 are required for cell migration and axon guidance. However, the roles in cell migration of all of the Caenorhabditis elegans Wnt genes and their candidate receptors have not been explored fully. We have extended our analysis to include all C. elegans Wnts and six candidate Wnt receptors: four Frizzleds, the sole Ryk family receptor LIN-18, and the Ror receptor tyrosine kinase CAM-1. We show that three of the Wnts, CWN-1, CWN-2, and EGL-20, play major roles in directing cell migrations and that all five Wnts direct specific cell migrations either by acting redundantly or by antagonizing each other's function. We report that all four Frizzleds function to direct Q-descendant cell migrations, but only a subset of the putative Wnt receptors function in directing migrations of other cells. Finally, we find striking differences between the phenotypes of the Wnt quintuple and Frizzled quadruple mutants.     

UNC-71, a disintegrin and metalloprotease (ADAM) protein,regulates motor axon guidance and sex myoblast migration in C. elegans

The migration of cells and growth cones is a process that is guided by extracellular cues and requires the controlled remodeling of the extracellular matrix along the migratory path. The ADAM proteins are important regulators of cellular adhesion and recognition because they can combine regulated proteolysis with modulation of cell adhesion. We report that the C. elegans gene unc-71 encodes a unique ADAM with an inactive metalloprotease domain. Loss-of-function mutations in unc-71 cause distinct defects in motor axon guidance and sex myoblast migration. Many unc-71 mutations affect the disintegrin and the cysteine-rich domains, supporting a major function of unc-71 in cell adhesion. UNC-71 appears to be expressed in a selected set of cells. Genetic mosaic analysis and tissue-specific expression studies indicate that unc-71 acts in a cell non-autonomous manner for both motor axon guidance and sex myoblast migration. Finally, double mutant analysis of unc-71 with other axon guidance signaling molecules suggests that UNC-71 probably functions in a combinatorial manner with integrins and UNC-6/netrin to provide distinct axon guidance cues at specific choice points for motoneurons.     

Syndecan regulates cell migration and axon guidance in C. elegans

During nervous system development, axons that grow out simultaneously in the same extracellular environment are often sorted to different target destinations. As there is only a restricted set of guidance cues known, regulatory mechanisms are likely to play a crucial role in controlling cell migration and axonal pathfinding. Heparan sulfate proteoglycans (HSPGs) carry long chains of differentially modified sugar residues that have been proposed to encode specific information for nervous system development. Here, we show that the cell surface proteoglycan syndecan SDN-1 functions autonomously in neurons to control the neural migration and guidance choices of outgrowing axons. Epistasis analysis suggests that heparan sulfate (HS) attached to SDN-1 can regulate guidance signaling by the Slit/Robo pathway. Furthermore, SDN-1 acts in parallel with other HSPG core proteins whose HS side chains are modified by the C5-epimerase HSE-5, and/or the 2O-sulfotransferase HST-2, depending on the cellular context. Taken together, our experiments show that distinct HS modification patterns on SDN-1 are involved in regulating axon guidance and cell migration in C. elegans.     

Axon guidance: asymmetric signaling orients polarized outgrowth

A network of connections is established as neural circuits form between neurons. To make these connections, neurons initiate asymmetric axon outgrowth in response to extracellular guidance cues. Within the specialized growth cones of migrating axons, F-actin and microtubules asymmetrically accumulate where an axon projects forward. Although many guidance cues, receptors and intracellular signaling components that are required for axon guidance have been identified, the means by which the asymmetry is established and maintained is unclear. Here, we discuss recent studies in invertebrate and vertebrate organisms that define a signaling module comprising UNC-6 (the Caenorhabditis elegans ortholog of netrin), UNC-40 (the C. elegans ortholog of DCC), PI3K, Rac and MIG-10 (the C. elegans ortholog of lamellipodin) and we consider how this module could establish polarized outgrowth in response to guidance cues.     

Navigating the cell

Changes in cell shape are associated with a variety of processes including cell migration, axon outgrowth, cell division, and vesicle trafficking. C. elegans UNC-53 and its vertebrate homologs, the Navigators, are required for the migration of cells and the outgrowth of neuronal processes. The identification of novel molecular interactions and live imaging studies have revealed that UNC-53/NAVs are signal transducers associated with actin filaments, microtubules, and intermediate filaments. In addition to modulating cytoskeletal dynamics at the leading edge of migrating or outgrowing cells, both UNC-53 and the navigators are expressed in adult cells, conspicuously those with specialized roles in endocytosis or secretion. Collectively, these results suggest that UNC-53/NAVs may be a central regulator of cytoskeletal dynamics, responsible for integrating signaling cues to multiple components of the cytoskeleton to coordinate rearrangement during cell outgrowth or trafficking.     

RACK-1 acts with Rac GTPase signaling and UNC-115/abLIM in Caenorhabditis elegans axon pathfinding and cell migration

Migrating cells and growth cones extend lamellipodial and filopodial protrusions that are required for outgrowth and guidance. The mechanisms of cytoskeletal regulation that underlie cell and growth cone migration are of much interest to developmental biologists. Previous studies have shown that the Arp2/3 complex and UNC-115/abLIM act redundantly to mediate growth cone lamellipodia and filopodia formation and axon pathfinding. While much is known about the regulation of Arp2/3, less is known about regulators of UNC-115/abLIM. Here we show that the Caenorhabditis elegans counterpart of the Receptor for Activated C Kinase (RACK-1) interacts physically with the actin-binding protein UNC-115/abLIM and that RACK-1 is required for axon pathfinding. Genetic interactions indicate that RACK-1 acts cell-autonomously in the UNC-115/abLIM pathway in axon pathfinding and lamellipodia and filopodia formation, downstream of the CED-10/Rac GTPase and in parallel to MIG-2/RhoG. Furthermore, we show that RACK-1 is involved in migration of the gonadal distal tip cells and that the signaling pathways involved in this process might be distinct from those involved in axon pathfinding. In sum, these studies pinpoint RACK-1 as a component of a novel signaling pathway involving Rac GTPases and UNC-115/abLIM and suggest that RACK-1 might be involved in the regulation of the actin cytoskeleton and lamellipodia and filopodia formation in migrating cells and growth cones.     

Distinct rac activation pathways control Caenorhabditis elegans cell migration and axon outgrowth

Rac GTPases act as molecular switch in various morphogenic events. However, the regulation of their activities during the development of multicellular organisms is not well understood. Caenorhabditis elegans rac genes ced-10 and mig-2 have been shown to act redundantly to control P cell migration and the axon outgrowth of D type motoneurons. We showed that ced-10 and mig-2 also control amphid axon outgrowth and amphid dendrite fasciculation in a redundant fashion. Our biochemical and genetic data indicate that unc-73, which encodes a protein related to Trio-like guanine nucleotide exchange factor, acts as a direct activator of ced-10 and mig-2 during P cell migration and axon outgrowth of D type motoneurons and amphid sensory neurons. Furthermore, rac regulators ced-2/crkII and ced-5/dock180 function genetically upstream of ced-10 and mig-2 during axon outgrowth of D type motoneurons and act upstream of mig-2 but not ced-10 during P cell migration. However, neither ced-2/crkII nor ced-5/dock180 is involved in amphid axon outgrowth. Therefore, distinct rac regulators control ced-10 and mig-2 differentially in various cellular processes.     

To move or not to move? Semaphorin signalling in cell migration

Semaphorins were discovered 11 years ago as molecular cues for axon guidance that are conserved from invertebrates to humans. More than 20 semaphorin genes have been identified in mammals and their protein products are now known to be involved in a range of processes from the guidance of cell migration to the regulation of the immune response, angiogenesis and cancer. Plexins, either alone or in association with neuropilins, constitute high-affinity semaphorin receptors. However, other transmembrane molecules have been implicated in semaphorin receptor complexes, and interactions between plexins and a range of intracellular effectors have been reported. These data indicate that semaphorins might be able to elicit responses through more than one signalling pathway. Interestingly, according to recent findings, the semaphorin-dependent control of cell migration crucially involves integrin-based adhesive structures through which polarized cell-membrane protrusion is coupled to cytoskeletal dynamics. This review focuses on the mechanisms whereby semaphorins are thought to regulate cell migration.     

The Caenorhabditis elegans Eph receptor activates NCK and N-WASP, and inhibits Ena/VASP to regulate growth cone dynamics during axon guidance

The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.     

Migration of neuronal cells along the anterior-posterior body axis of C. elegans: Wnts are in control

Migrating neuronal cells are directed to their final positions by an array of guidance cues. It has been shown that guidance molecules such as UNC-6/Netrin and SLT-1/Slit play a major role in controlling cell and axon migrations along the dorsal-ventral body axis. Much less is known, however, about the mechanisms that mediate migration along the anterior-posterior (AP) body axis. Recent research in Caenorhabditis elegans has uncovered an important role of the Wnt family of signalling molecules in controlling AP-directed neuronal cell migration and polarity. A common theme that emerges from these studies is that multiple Wnt proteins function in parallel as instructive cues or permissive signals to control neuronal patterning along this major body axis.     

The NC1/endostatin domain of Caenorhabditis elegans type XVIII collagen affects cell migration and axon guidance

Type XVIII collagen is a homotrimeric basement membrane molecule of unknown function, whose COOH-terminal NC1 domain contains endostatin (ES), a potent antiangiogenic agent. The Caenorhabditis elegans collagen XVIII homologue, cle-1, encodes three developmentally regulated protein isoforms expressed predominantly in neurons. The CLE-1 protein is found in low amounts in all basement membranes but accumulates at high levels in the nervous system. Deletion of the cle-1 NC1 domain results in viable fertile animals that display multiple cell migration and axon guidance defects. Particular defects can be rescued by ectopic expression of the NC1 domain, which is shown to be capable of forming trimers. In contrast, expression of monomeric ES does not rescue but dominantly causes cell and axon migration defects that phenocopy the NC1 deletion, suggesting that ES inhibits the promigratory activity of the NC1 domain. These results indicate that the cle-1 NC1/ES domain regulates cell and axon migrations in C. elegans.     

Plexins, semaphorins, and scatter factor receptors: a common root for cell guidance signals?

Semaphorins, the plexin family of semaphorin receptors, and scatter factor receptors share evolutionarily conserved protein modules, such as the semaphorin domain and Met Related Sequences (MRS). All these proteins also have in common a role in mediating cell guidance cues. During development, scatter factor receptors control cell migration, epithelial tubulogenesis, and neurite extension. Semaphorins and their receptors are known signals for axon guidance; they are also suspected to regulate developmental processes involving cell migration and morphogenesis, and have been implicated in immune function and tumor progression. Scatter factors and secreted semaphorins are diffusible ligands, whereas membrane-bound semaphorins signal by cell-cell interaction. Cell guidance control by semaphorins requires plexins, alone or in a receptor complex with neuropilins. Semaphorins, besides their role in axon guidance, are expected to have multiple functions in morphogenesis and tissue remodeling by mediating cell-repelling cues through plexin receptors.     

Three C. elegans Rac proteins and several alternative Rac regulators control axon guidance, cell migration and apoptotic cell phagocytosis.

The Caenorhabditis elegans genome contains three rac-like genes, ced-10, mig-2, and rac-2. We report that ced-10, mig-2 and rac-2 act redundantly in axon pathfinding: inactivating one gene had little effect, but inactivating two or more genes perturbed both axon outgrowth and guidance. mig-2 and ced-10 also have redundant functions in some cell migrations. By contrast, ced-10 is uniquely required for cell-corpse phagocytosis, and mig-2 and rac-2 have only subtle roles in this process. Rac activators are also used differentially. The UNC-73 Trio Rac GTP exchange factor affected all Rac pathways in axon pathfinding and cell migration but did not affect cell-corpse phagocytosis. CED-5 DOCK180, which acts with CED-10 Rac in cell-corpse phagocytosis, acted with MIG-2 but not CED-10 in axon pathfinding. Thus, distinct regulatory proteins modulate Rac activation and function in different developmental processes.     

Eph受体家族及其配体的信号转导途径及功能

Eph受体是已知最大的酪氨酸蛋白激酶受体家族,Eph受体和其膜附着型配体（ephrin）在发育过程中呈现不同的表达模式,近来研究证明,Eph受体和其配体在包括神经网络形成,神经管和轴旁中胚层的成型（patterning）,细胞迁移导向和轴突路径导引,血管形成等许多的发育过程中起重要作用.Eph受体及其配体也与肿瘤发生有关,因此深入分析这些分子尤其在肿瘤细胞生长中的功能而应用于治疗具有重要的临床意义.     

Dissection of cell division processes in the one cell stage Caenorhabditis elegans embryo by mutational analysis

To identify novel components required for cell division processes in complex eukaryotes, we have undertaken an extensive mutational analysis in the one cell stage Caenorhabditis elegans embryo. The large size and optical properties of this cell permit observation of cell division processes with great detail in live specimens by simple differential interference contrast (DIC) microscopy. We have screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III with time-lapse DIC video microscopy. Using this assay, we have identified 48 mutations in 34 loci which are required for specific cell division processes in the one cell stage embryo. We show that mutations fall into distinct phenotypic classes which correspond, among others, to the processes of pronuclear migration, rotation of centrosomes and associated pronuclei, spindle assembly, chromosome segregation, anaphase spindle positioning, and cytokinesis. We have further analyzed pronuclear migration mutants by indirect immunofluorescence microscopy using antibodies against tubulin and ZYG-9, a centrosomal marker. This analysis revealed that two pronuclear migration loci are required for generating normal microtubule arrays and four for centrosome separation. All 34 loci have been mapped by deficiencies to distinct regions of chromosome III, thus paving the way for their rapid molecular characterization. Our work contributes to establishing the one cell stage C. elegans embryo as a powerful metazoan model system for dissecting cell division processes.     

Interactions of UNC-34 Enabled with Rac GTPases and the NIK kinase MIG-15 in Caenorhabditis elegans axon pathfinding and neuronal migration

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 pathfinding. 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.