Netrin-A and Netrin-B


Planarian Netrin homologs

Conserved axon guidance mechanisms are essential for proper wiring of the nervous system during embryogenesis; however, the functions of these cues in adults and during regeneration remain poorly understood. Because freshwater planarians can regenerate a functional central nervous system (CNS) from almost any portion of their body, they are useful models in which to study the roles of guidance cues during neural regeneration. Two netrin homologs and one netrin receptor family member were characterized from Schmidtea mediterranea. RNAi analyses indicate that Smed-netR (netrin receptor) and Smed-netrin2 are required for proper CNS regeneration and that Smed-netR may mediate the response to Smed-netrin2. Remarkably, Smed-netR and Smed-netrin2 are also required in intact planarians to maintain the proper patterning of the CNS. These results suggest a crucial role for guidance cues, not only in CNS regeneration but also in maintenance of neural architecture (Cebria, 2005).

UNC-6, a C. elegans Netrin homolog

Netrins are laminin-related proteins that guide circumferential migrations on the ectoderm. To understand how netrin cues direct cell movements, the expression of nematode netrin UNC-6 has been examined at all stages from embryo to adult. UNC-6 is expressed in 12 types of neuroglia and neurons, creating a hierarchy of netrin cues in the developing nervous system. Multiple netrin cues, each with a characteristic role, guide cells and axons during development. The biological activities of netrins are mediated through distinct protein domains. Subtle mutations in domain VI can produce selective defects in both direction- and tissue-specific guidance. EGF-like module V-2 is essential for dorsal guidance activity; this module is important for interactions between UNC-6 and the dorsal guidance receptor UNC-5 (Wadsworth, 1996).

The netrin guidance cue, UNC-6, and the netrin receptors, UNC-5 and UNC-40 (Drosophila homolog: Frazzled), guide SDQR cell and axon migrations in C. elegans. This neuron is part of the sublateral nerves that develop from axons of embryonic neurons in the ring ganglia that run either anteriorly or posteriorly along the epidermis beneath the four body wall muscles. The posteriorly directed sublateral nerves, in particular, comprise the axons from neurons SIA, SIB and SMB in the ventral ganglion, plus SMD in the lateral ganglia. During the first larval stage, the axons of postembryonic neurons SDQ and PLN join the dorsal anterior and ventral posterior sublateral nerves, respectively. Shortly after its birth, the SDQR neuron migrates dorsally to the lateral margin of the body wall muscle alongside the ALMR associated nerve. Insinuating beneath the body wall muscle and epidermis, the SDQR axon grows dorsally and then anteriorly along the dorsal epidermis toward the nerve ring. SDQR provides several advantages for study: (1) the cell and axon are relatively isolated from surrounding neurons; (2) the cell is born in the first larval stage, after the final expression pattern of UNC-6 has occurred; (3) the sister neuron of SDQR, AVM, migrates ventrally to a position that is slightly posterior to SDQR. This neuron serves as an easily recognizable positional reference and as a control for SDQR precursor cell migration and cell lineage (Kim, 1999).

In wild-type larvae, SDQR migrations are away from ventral UNC-6-expressing cells, suggesting that UNC-6 repels SDQR. In unc-6 null larvae, SDQR migrations are toward the ventral midline, indicating a response to other guidance cues that directs the migrations ventrally. Although ectopic UNC-6 expression dorsal to the SDQR cell body would be predicted to cause ventral SDQR migrations in unc-6 null larvae, in fact, more migrations are directed dorsally, suggesting that SDQR is not always repelled from the dorsal source of UNC-6. UNC-5 is required for dorsal SDQR migrations, but not for the ventral migrations in unc-6 null larvae. UNC-40 appears to moderate both the response to UNC-6 and to the other cues. These results show that SDQR responds to multiple guidance cues and they suggest that, besides UNC-6, other factors influence whether an UNC-6 responsive cell migrates toward or away from an UNC-6 source in vivo. It is proposed that multiple signals elicited by the guidance cues are integrated and interpreted by SDQR and that the response to UNC-6 can change depending on the combination of cues encountered during migration (Kim, 1999).

A model is provided for cues that position SDQR dorsoventrally. The UNC-6 guidance cue is distributed in a ventral to dorsal gradient along the body wall. A second cue(s) is localized to the dorsal sublateral region. During the initial SDQR cell and axon migration, receptor complexes on SDQR mediate a repulsive response to UNC-6 that guides SDQR dorsally. SDQR interaction with the dorsal sublateral cue causes the response to UNC-6 to be modified so that SDQR is no longer repelled. Within this zone, the SDQR axon migrates to the anterior. A third cue can attract UNC-6 ventrally only in the absence of UNC-6. Epidermal cells and muscle cells could be sources of localized cues. These responses determine the final dorsoventral position of the SDQR cell and axon (Kim, 1999).

In the Caenorhabditis elegans embryo, some ventral midline motoneurons extend a process circumferentially to the dorsal midline and a process longitudinally along ventral nerve cord interneurons. Circumferential migrations are guided by netrin UNC-6, which repels motoneuron axons dorsally. Although the motoneuron cell bodies and the longitudinal axons are positioned along UNC-6-expressing interneurons in the ventral nerve cord, the circumferential processes extend only from the motoneuron cell bodies and from defined locations along some longitudinal axons. This implies a mechanism that regulates motoneuron branching of UNC-6-responsive processes. UNC-6 is a laminin-related protein of 591 amino acids. Domains comprising residues 1-437 are homologous to the N-terminal domains VI, V-1, V-2, and V-3 of laminin subunits, whereas the domain comprising residues 438-591, designated domain C, has no homology to laminin subunits. The 153 residue C terminus is shared among netrins and is related to the C terminus of Frzb, an antagonist of the Wnt receptor. The direction- and tissue-specific guidance activities are mediated by distinct domains of UNC-6. Within domain VI, one mutation, ev436, can produce selective defects in mesoblast but not axonal migrations, whereas another allele, ev437, selectively disrupts ventral migrations of both axons and mesoblasts. Four alleles that selectively cause defects in dorsal migrations each produce a protein that specifically lacks the V-2 epidermal growth factor-like module. Expression of unc-6DeltaC, which encodes UNC-6 without domain C, partially rescues circumferential migration defects in unc-6 null animals. This activity depends on the netrin receptors UNC-5 and UNC-40. These results indicate that UNC-6DeltaC can provide the circumferential guidance functions of UNC-6. Expression of unc-6DeltaC causes motoneuron branching and the extension of processes from abnormal positions along the ventral nerve cord. This activity is also UNC-5- and UNC-40-dependent. It is proposed that local interactions mediated by domain C regulate motoneuron branching and responsiveness to the UNC-6 cue (Lim, 1999).

The DA, DB, and DD motoneuron cell bodies are arranged in a row along the ventral midline. In the mature animal, the two fascicles of the ventral nerve cord flank the motoneuron cell bodies. The early development of the ventral nerve cord and the outgrowth of the motoneuron processes have been described from electron microscope serial reconstructions. Initially, the AVG axon migrates posteriorly from an anterior midline cell body and pioneers the right side of the ventral cord. This is followed by DD processes extending forward along the AVG axon. Soon after, processes from all three classes of motoneurons simultaneously migrate dorsally. The processes of DA and DB motoneurons extend from the cell bodies, whereas the processes from DD motoneurons extend from near the anterior ends of their axons. Interestingly, the DD motoneuron circumferential extensions are always directly opposite DB cell bodies, suggesting that proximity to the DB cell body influences the point of outgrowth. After the motoneuron processes reach the dorsal midline and begin to form the dorsal nerve cord, additional interneuron axons from cell bodies in the head migrate along the ventral nerve cord. At the same time, from each DA and DB cell body a second process grows out along the ventral nerve cord (Lim, 1999).

Several guidance cues must work in concert to direct motoneuron axon migrations. Interestingly, the ventral nerve cord interneurons express several guidance cues that the motoneuron processes are responsive to. Fasciculation cues of the AVG pioneer and the later interneurons that innervate DA and DB to complete the motor circuitry are predicted to attract and guide the migrations longitudinally in the developing nerve cord. In addition, AVG and the later interneurons also express UNC-6 (Lim, 1999).

Domain C determines whether motoneuron processes migrate longitudinally along the interneurons in the ventral nerve cord or whether they branch and extend processes circumferentially in response to UNC-6. In wild-type animals, UNC-6-responsive processes branch and extend dorsally only at the motoneuron cell bodies and at defined positions along some of the longitudinal processes. Because the motoneurons are positioned along UNC-6-expressing interneurons, some mechanism must regulate where the branching occurs. In unc-6DeltaC animals, extra processes extend from the ventral nerve cord and are guided dorsally by UNC-6DeltaC. A simple model is that domain C mediates an activity at the ventral midline that prevents motoneuron processes from branching and responding to UNC-6. During the period of pioneer circumferential migrations in wild-type embryos, branching could be induced at the DA and DB motoneurons by molecules that block this activity (Lim, 1999).

It is proposed that multimeric receptor complexes on the surfaces of the interneurons and motoneuron axons control the balance between axon-axon adhesion and repulsion by UNC-6. UNC-5 and UNC-40 are components of these complexes, because unc-5 and unc-40 null mutations can suppress the branching response of the motoneurons to UNC-6DeltaC. Oligomerization of UNC-6 by the binding of domain C to interneuron-associated proteoglycans could help aggregate and activate the receptor complexes. Besides promoting axon-axon adhesion, these complexes could prevent UNC-5 and UNC-40 from mediating a repulsive response to UNC-6. In contrast, UNC-6DeltaC, which could not promote receptor aggregation, could interact with UNC-5 and UNC-40 and elicit repulsive and branching signals (Lim, 1999).

If domain C helps tether UNC-6 at interneuron surfaces, this model predicts that UNC-6DeltaC might diffuse further from the ventral nerve cord. However, this difference by itself does not account for the ventral cord phenotypes of unc-6DeltaC animals. (1) The diffusion of UNC-6DeltaC would be predicted to cause the peak of the repellent gradient to be reduced at the interneurons, and this should favor migration along the interneurons rather than repulsion from them. (2) Other experiments show that altering the distribution of UNC-6 does not induce branching and the extension of additional processes from the ventral nerve. For example, these phenotypes are not observed in unc-6 null mutants or in animals in which UNC-6 is ectopically expressed either from the touch receptor neurons or from neurons throughout the nervous system. Furthermore, the distribution of UNC-6DeltaC is probably not entirely different from that of UNC-6 in wild-type animals, because circumferential migrations are guided by the expression of unc-6DeltaC. Also, in any given unc-6DeltaC animal, the longitudinal motoneuron processes will migrate dorsally without preference for one side of the animal, suggesting that the peak of the repellent gradient has not been shifted to one side of the ventral cord. The normal circumferential processes keep their preference in unc-6DeltaC animals. Together these observations suggest that the instructive properties of the UNC-6DeltaC gradient are similar to those of the UNC-6 gradient in wild-type animals (Lim, 1999).

The motoneuron processes that have been induced to leave the ventral nerve cord by unc-6DeltaC expression migrate dorsally along either side of the body wall. At the boundary between the ventral sublateral and lateral body wall, these motoneuron axons either turn or they terminate. This suggests that inhibitory molecules associated with the lateral epidermis surface or with the lateral basement membrane prevent migration across the lateral epidermis. The ventral sublateral-lateral boundary is demarcated by the junction of ventral and lateral epidermal cells. In addition to encountering a different cellular substrate, the axons also encounter distinct basement membranes. It is speculated that the unique appearance and trajectories of these axons are because they are simultaneously directed dorsally by UNC-6DeltaC, despsite being inhibited from migrating across the lateral epidermis by cues associated with the lateral body wall. This further suggests that the motoneuron axons that normally migrate circumferentially have specific properties that allow them to migrate to the dorsal midline. These properties may only be conferred on the axons that extend during the normal period of circumferential migrations and not on axons that extend in response to UNC-6DeltaC at other times (Lim, 1999).

UNC-6DeltaC acts as an antagonist to the action of domain C at the ventral nerve cord. In wild-type animals, the ventral cord phenotype can be induced by the expression of the urIs77 transgene, suggesting that UNC-6DeltaC dominantly interferes with endogenous UNC-6. Furthermore, recent genetic screens have isolated suppressors of the unc-6DeltaC ventral cord phenotype that do not effect unc-6 guidance activities, indicating that this new activity is genetically separable from UNC-6 circumferential guidance activities. Finally, these results provide evidence that in vivo the influence of guidance cues can be modified locally to alter axon trajectories. By regulating spatial and temporal expression patterns and by locally modifying activities, a few guidance cues could direct the development of complex axon scaffolds (Lim, 1999).

Cell migrations play a critical role in animal development and organogenesis. Here, a mechanism is described by which the migration behaviour of a particular cell type is regulated temporally and coordinated with over-all development of the organism. The hermaphrodite distal tip cells (DTCs) of C. elegans migrate along the body wall in three sequential phases that can be distinguished by the orientation of their movements, which alternate between the anteroposterior and dorsoventral axes. The ventral-to-dorsal second migration phase requires the UNC-6 netrin guidance cue and its receptors UNC-5 and UNC-40, as well as additional UNC-6-independent guidance systems. Evidence is provided that the transcriptional upregulation of unc-5 in the DTCs is coincident with the initiation of the second migration phase, and that premature UNC-5 expression in these cells induces precocious turning in an UNC-6-dependent manner. The DAF-12 steroid hormone receptor, which regulates developmental stage transitions in C. elegans, is required for initiating the first DTC turn and for coincident unc-5 upregulation. Evidence is also presented for the existence of a mechanism that opposes or inhibits UNC-5 function during the longitudinal first migration phase and for a mechanism that facilitates UNC-5 function during turning. The facilitating mechanism presumably does not involve transcriptional regulation of unc-5 but may represent an inhibition of the phase 1 mechanism that opposes or inhibits UNC-5. These results, therefore, reveal the existence of two mechanisms that regulate the UNC-5 receptor and are critical for responsiveness to the UNC-6 netrin guidance cue and for linking the directional guidance of migrating distal tip cells to developmental stage advancements (Su, 2000).

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. 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. An interaction between MIG-10 and UNC-34, a protein that promotes actin-filament extension, is important in the response to guidance cues and MIG-10 colocalizes with actin in cultured cells, where it can induce the formation of lamellipodia. It is concluded 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 (Quinn, 2006).

A model predicts that MIG-10 activity is involved in the process of polarized axon growth. Although it is difficult to directly measure whether MIG-10 activity is polarized in C. elegans neurons, MIG-10 can induce lamellipodia in cultured cells. The asymmetric formation of lamellipodia has been implicated as an initial step toward polarized axon growth. The observations are also consistent with those described for two vertebrate MIG-10 orthologs, RIAM and lamellipodin. Overexpression of RIAM induces cell spreading and lamellipodia formation, and overexpression of lamellipodin has been shown to increase the velocity of actin-based protrusive activity in fibroblasts. RIAM interacts with Rap1, Ena/vasp proteins, and profilin [14]. Lamellipodin is localized to the tips of filopodia in fibroblasts and neuronal growth cones and can interact with Ena/vasp proteins. Interestingly, lamellipodin can interact with PI(3,4) phosphoinostide, a molecule that is asymmetrically localized in response to chemotactic cues in several types of . The ability of lamellipodin to interact with an asymmetrically localized molecule suggests a potential mechanism that could mediate the polarization of MIG-10 activity in response to guidance cues (Quinn, 2006).

The cytoplasmic C. elegans protein MIG-10 affects cell migrations and is related to mammalian proteins that bind phospholipids and Ena/VASP actin regulators. In cultured cells, mammalian MIG-10 promotes lamellipodial growth and Ena/VASP proteins induce filopodia. This study shows that during neuronal development, mig-10 and the C. elegans Ena/VASP homolog unc-34 cooperate to guide axons toward UNC-6 (netrin) and away from SLT-1 (Slit). The single mutants have relatively mild phenotypes, but mig-10; unc-34 double mutants arrest early in development with severe axon guidance defects. In axons that are guided toward ventral netrin, unc-34 is required for the formation of filopodia and mig-10 increases the number of filopodia. In unc-34 mutants, developing axons that lack filopodia are still guided to netrin through lamellipodial growth. In addition to its role in axon guidance, mig-10 stimulates netrin-dependent axon outgrowth in a process that requires the age-1 phosphoinositide-3 lipid kinase but not unc-34. It is concluded that mig-10 and unc-34 organize intracellular responses to both attractive and repulsive axon guidance cues. mig-10 and age-1 lipid signaling promote axon outgrowth; unc-34 and to a lesser extent mig-10 promote filopodia formation. Surprisingly, filopodia are largely dispensable for accurate axon guidance (Chang, 2006).

Polarity is an essential feature of many cell types, including neurons that receive information from local inputs within their dendrites and propagate nerve impulses to distant targets through a single axon. It is generally believed that intrinsic structural differences between axons and dendrites dictate the polarized localization of axonal and dendritic proteins. However, whether extracellular cues also instruct this process in vivo has not been explored. This study shows that the axon guidance cue UNC-6/netrin and its receptor UNC-5 act throughout development to exclude synaptic vesicle and active zone proteins from the dendrite of the C. elegans motor neuron DA9, which is proximal to a source of UNC-6/netrin. In unc-6/netrin and unc-5 loss-of-function mutants, presynaptic components mislocalize to the DA9 dendrite. In addition, ectopically expressed UNC-6/netrin, acting through UNC-5, is sufficient to exclude endogenous synapses from adjacent subcellular domains within the DA9 axon. Furthermore, this anti-synaptogenic activity is interchangeable with that of LIN-44/Wnt despite being transduced through different receptors, suggesting that extracellular cues such as netrin and Wnts not only guide axon navigation but also regulate the polarized accumulation of presynaptic components through local exclusion (Poon, 2009).

The UNC-6/netrin guidance cue functions in axon guidance in vertebrates and invertebrates, mediating attraction via UNC-40/DCC family receptors and repulsion via by UNC-5 family receptors. The growth cone reads guidance cues and extends lamellipodia and filopodia, actin-based structures that sense the extracellular environment and power the forward motion of the growth cone. This study shows that UNC-6/netrin, UNC-5 and UNC-40/DCC modulate the extent of growth cone protrusion that correlated with attraction versus repulsion. Loss-of-function unc-5 mutants display increased protrusion in repelled growth cones, whereas loss-of-function unc-6 or unc-40 mutants causes decreased protrusion. In contrast to previous studies, this work suggests that the severe guidance defects in unc-5 mutants may be due to latent UNC-40 attractive signaling that steers the growth cone back towards the ventral source of UNC-6. UNC-6/Netrin signaling also controls polarity of growth cone protrusion and F-actin accumulation that correlates with attraction versus repulsion. However, filopodial dynamics are affected independently of polarity of protrusion, indicating that the extent versus polarity of protrusion are at least in part separate mechanisms. In summary, this study showa that growth cone guidance in response to UNC-6/netrin involves a combination of polarized growth cone protrusion as well as a balance between stimulation and inhibition of growth cone (e.g. filopodial) protrusion (Norris, 2011).

Synaptic vesicle clustering requires a distinct MIG-10/Lamellipodin isoform and ABI-1 downstream from Netrin

The chemotrophic factor Netrin can simultaneously instruct different neurodevelopmental programs in individual neurons in vivo. How neurons correctly interpret the Netrin signal and undergo the appropriate neurodevelopmental response is not understood. This study identified MIG-10 isoforms as critical determinants of individual cellular responses to Netrin. Distinct MIG-10 isoforms, varying only in their N-terminal motifs, can localize to specific subcellular domains and are differentially required for discrete neurodevelopmental processes in vivo. MIG-10B was identified as an isoform uniquely capable of localizing to presynaptic regions and instructing synaptic vesicle clustering in response to Netrin. MIG-10B interacts with Abl-interacting protein-1 (ABI-1)/Abi1, a component of the WAVE complex, to organize the actin cytoskeleton at presynaptic sites and instruct vesicle clustering through SNN-1/Synapsin. A motif in the MIG-10B N-terminal domain was identified that is required for its function and localization to presynaptic sites. With this motif, a dominant-negative MIG-10B construct was engineered that disrupts vesicle clustering and animal thermotaxis behavior when expressed in a single neuron in vivo. These findings indicate that the unique N-terminal domains confer distinct MIG-10 isoforms with unique capabilities to localize to distinct subcellular compartments, organize the actin cytoskeleton at these sites, and instruct distinct Netrin-dependent neurodevelopmental programs (Stavoe, 2012).

MIG-10 functions with ABI-1 to mediate the UNC-6 and SLT-1 axon guidance signaling pathways

Extracellular guidance cues steer axons towards their targets by eliciting morphological changes in the growth cone. A key part of this process is the asymmetric recruitment of the cytoplasmic scaffolding protein MIG-10 (lamellipodin). MIG-10 is thought to asymmetrically promote outgrowth by inducing actin polymerization. However, the mechanism that links MIG-10 to actin polymerization is not known. This study identified the actin regulatory protein ABI-1 as a partner for MIG-10 that can mediate its outgrowth-promoting activity. The SH3 domain of ABI-1 binds to MIG-10, and loss of function of either of these proteins causes similar axon guidance defects. Like MIG-10, ABI-1 functions in both the attractive UNC-6 (netrin) pathway and the repulsive SLT-1 (slit) pathway. Dosage sensitive genetic interactions indicate that MIG-10 functions with ABI-1 and WVE-1 to mediate axon guidance. Epistasis analysis reveals that ABI-1 and WVE-1 function downstream of MIG-10 to mediate its outgrowth-promoting activity. Moreover, experiments with cultured mammalian cells suggest that the interaction between MIG-10 and ABI-1 mediates a conserved mechanism that promotes formation of lamellipodia. Together, these observations suggest that MIG-10 interacts with ABI-1 and WVE-1 to mediate the UNC-6 and SLT-1 guidance pathways (Xu, 2012).

Conserved and novel functions for Netrin in the formation of the axonal scaffold and glial sheath cells in spiders

Netrins are well known for their function as long-range chemotropic guidance cues, in particular in the ventral midline of vertebrates and invertebrates. Over the past years, publications are accumulating that support an additional short-range function for Netrins in diverse developmental processes such as axonal pathfinding and cell adhesion. This study describes the formation of the axonal scaffold in the spiders Cupiennius salei and Achaearanea tepidariorum and shows that axonal tract formation seems to follow the same sequence as in insects and crustaceans in both species. First, segmental neuropiles are established which then become connected by the longitudinal fascicles. Interestingly, the commissures are established at the same time as the longitudinal tracts despite the large gap between the corresponding hemi-neuromeres which results from the lateral movement of the germband halves during spider embryogenesis. Netrin has a conserved function in the ventral midline in commissural axon guidance. This function is retained by an adaptation of the expression pattern to the specific morphology of the spider embryo. Furthermore, a novel function is demonstrated of netrin in the formation of glial sheath cells that has an impact on neural precursor differentiation. Loss of Netrin function leads to the absence of glial sheath cells which in turn results in premature segregation of neural precursors and overexpression of the early motor- and interneuronal marker islet. It is suggested that Netrin is required in the differentiated sheath cells for establishing and maintaining the interaction between NPGs and sheath cells. This short-range adhesive interaction ensures that the neural precursors maintain their epithelial character and remain attached to the NPGs. Both the conserved and novel functions of Netrin seem to be required for the proper formation of the axonal scaffold (Linne, 2011).

Netrin in the sea anemone

Nearly all metazoans show signs of bilaterality, yet it is believed the bilaterians arose from radially symmetric forms hundreds of millions of years ago. Cnidarians (corals, sea anemones, and 'jellyfish') diverged from other animals before the radiation of the Bilateria. They are diploblastic and are often characterized as being radially symmetrical around their longitudinal (oral-aboral) axis. The deployment of orthologs of a number of family members of developmental regulatory genes that are expressed asymmetrically during bilaterian embryogenesis from the sea anemone, Nematostella vectensis, have been studied. The secreted TGF-beta genes Nv-dpp, Nv-BMP5-8, six TGF-beta antagonists (NvChordin, NvNoggin1, NvNoggin2, NvGremlin, NvFollistatin, and NvFollistatin-like), the homeodomain proteins NvGoosecoid (NvGsc) and NvGbx, and the secreted guidance factor, NvNetrin, were studied. NvDpp, NvChordin, NvNoggin1, NvGsc, and NvNetrin are expressed asymmetrically along the axis perpendicular to the oral-aboral axis, the directive axis. Furthermore, NvGbx, and NvChordin are expressed in restricted domains on the left and right sides of the body, suggesting that the directive axis is homologous with the bilaterian dorsal-ventral axis. The asymmetric expression of NvNoggin1 and NvGsc appear to be maintained by the canonical Wnt signaling pathway. The asymmetric expression of NvNoggin1, NvNetrin, and Hox orthologs NvAnthox7, NvAnthox8, NvAnthox1a, and NvAnthox6, in conjunction with the observation that NvNoggin1 is able to induce a secondary axis in Xenopus embryos argues that N. vectensis could possess antecedents of the organization of the bilaterian central nervous system (Matus, 2006).

Amphioxus netrin

The conserved function of netrins in triploblasts, coupled with the phylogenetic position of amphioxus (the closest living relative of the vertebrates), has been used to investigate the evolution of an axon guidance cue in chordates. A single amphioxus netrin gene was isolated by PCR and cDNA library screening and named AmphiNetrin. The predicted AmphiNetrin protein shows high identity to other netrin family members but differ in that the third of three EGF repeats found in other netrins is absent. Despite the absent EGF repeat AmphiNetrin is most closely related to the vertebrate netrins. AmphiNetrin is expressed in embryonic notochord and floor plate, a pattern similar to that of vertebrate netrin-1 expression. AmphiNetrin expression is also identified more widely in the posterior larval brain, and in the anterior extension of the notochord that underlies the anterior of the amphioxus brain. These areas of expression are correlated with developing axon trajectories: The floor plate with ventrally projecting somatic motor neurons and Rohde cell projections, the posterior brain with the ventral commissure and primary motor centre and the anterior extension of the notochord with ventrally projecting neurons associated with the median eye. Amphioxus is naturally cyclopaedic and also lacks ventral brain cells: the induction of these cells results in the splitting of the vertebrate eye field; when these cells are missing, the result is cyclopaedia. These cells normally express netrins required for developing axon tracts in the brain, and the expression of AmphiNetrin in the anterior extension of the notochord underlying the brain may explain how amphioxus is able to maintain ventral guidance cues while lacking these cells (Shimeld, 2000).

Cloning, expression and mutation of vertebrate netrins

Continued: Netrins Evolutionary homologs  part 2/3 |  part 3/3

Netrin-A and Netrin-B: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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