Frazzled

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-40, a C. elegans Frazzled homolog

The unc-40 gene of C. elegans shares domain structures with Frazzled, DCC and Neogenin. In the extracellular domain, UNC-40 is 30% identical to DCC and 26% identical to neogenin. In comparision, the vertebrate proteins are 49% identical to one another in this region. In the intracellular domain, UNC-40 is only 13% identical to DCC and 10% identical to neogenin. In comparision, the vertebrate proteins are 37% identical to one another. Despite their divergence, the intracellular domains of all three proteins are very similar in length and share at least one sequence motif, PXDLWI (residues 1142-47), also shared with Frazzled (Chan, 1996).

At the onset of gastrulation UNC-40 becomes detectable on the surface of all cells, and then gradually decreases. In the neurula, UNC-40 is highly expressed on ventral cord motorneurons undergoing axonogenesis, including cell bodies and axons. In first stage larva, certain ventral epidermoblasts express UNC-40 as they undergo planar movements within the epithelium. Similarly, neuroblasts QL and QR and their descendents express UNC-40 as they migrate longitudinally along the epidermis. In second stage and later larva, the distal tip cells of hermaphrodites express UNC-40 as they migrate along the body wall. UNC-40 is required in neurons responding to UNC-6 Netrin cues. (See also the Drosophila homologs to Netrin) In unc-40 mutants, axons from AVM and PVM neurons often fail to reach the ventral cord. UNC-40 acts cell-autonomously in orienting growth cones. UNC-40 has several developmental functions not requiring UNC-6. Generalized expression of UNC-40 at gastrulation, well before known UNC-6 functions could contribute to embryonic morphogenesis and elongation (Chan, 1996).

UNC-5, another C. elegans immunoglobulin superfamily member (see Drosophila unc-5), is also required for repulsive responses to Netrin, but unlike UNC-40 plays no role in attractive responses to netrins. Ectopic expression of UNC-5 in UNC-40 expressing neurons (whose axons normally grow toward UNC-6 sources such as AVM and PVM) is sufficient to reorient growth cones of these axons away from netrin sources. Thus UNC-5 is capable of conveying polarity information to the growth cone, presumably by mediating an aversive response to this netrin, and orienting it away from the high concentrations of UNC-6 that are present ventrally. This instructive function of UNC-5 requires the cooperation of UNC-40. Thus UNC-5 appears to act as a co-receptor. This work implies that the structure of the netrin receptor may involve more than one gene product (Chan, 1996 and references).

The UNC-5 guidance receptor, in response to the UNC-6/netrin path cue, orients growing axons in a dorsal direction along the epidermis of Caenorhabditis elegans. When ectopically expressed in the touch neurons, which normally extend ventrally or longitudinally, UNC-5 is able to reorient their axons toward the dorsal side in an UNC-6-dependent manner. This forms the basis of a genetic screen to identify other mutations that, like unc-6 mutations, suppress unc-5-induced growth cone guidance. These mutations may identify new components required for pioneer axon guidance by unc-5. This paper describes eight genes that are required for ectopic unc-5-induced growth cone steering. Mutations in four of these identify the previously known axon guidance genes [unc-6 (the ligand for UNC-5), unc-40 (ankyrin repeat proteins serving as a putative link between UNC-5 and the cytoskeleton), unc-34, and unc-44]; mutations in four others identify the novel genes unc-129, seu-1, seu-2, and seu-3. Several of these mutations cause axon guidance defects similar to those found in unc-5 mutants. It is proposed that some or all of these genes may function in a developmentally important unc-5 signaling pathway (Colavita, 1998).

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).

The bilateral C. elegans neuroblasts QL and QR are born in the same anterior/posterior (A/P) position, but polarize and migrate left/right asymmetrically: QL migrates toward the posterior and QR migrates toward the anterior. After their migrations, QL but not QR switches on the Hox gene mab-5. The UNC-40/netrin receptor and a novel transmembrane protein containing 13 hydrophobic domains, DPY-19, are required to orient these cells correctly. In unc-40 or dpy-19 mutants, the Q cells polarize randomly; in fact, an individual Q cell polarizes in multiple directions over time. In addition, either cell can express MAB-5. Both UNC-40 and DPY-19, as well as the Trio/GTPase exchange factor homolog UNC-73, are required for full polarization and migration. Thus, these proteins appear to participate in a signaling system that orients and polarizes these migrating cells in a left/right asymmetrical fashion during development. The C. elegans netrin UNC-6, which guides many cells and axons along the dorsoventral axis, is not involved in Q cell polarization, suggesting that a different netrin-like ligand serves to polarize these cells along the anteroposterior axis (Honigberg, 2000).

Previous studies suggest that QL but not QR activates MAB-5 expression during normal development because QL and QR have different response thresholds to EGL-20/Wnt, which is produced in the posterior body region near the tail. Mutations in unc-40 and dpy-19 randomize the L/R asymmetrical pattern of MAB-5 expression. The probability that a Q cell would express MAB-5 correlates with the relative amount of time spent pointing toward the posterior. Why should such a correlation exist? One possibility is that polarization towards the posterior increases the exposure of the cells to an activator of Wnt signaling that is located posterior to QL, or decreases its exposure to an anteriorly localized repressor. A second possibility is that polarization allows QL or QR to make a stable cell contact, which in turn regulates sensitivity to the EGL-20 signal. Such a model is attractive because cell contact is known to regulate an EGL-20/Wnt signaling pathway that can activate MAB-5 expression in the cells V5 and V6. Finally, unc-40 and dpy-19 could act at the head of a regulatory cascade that governs both Q cell polarization and EGL-20 sensitivity in parallel (Honigberg, 2000).

Netrins promote axon outgrowth and turning through DCC/UNC-40 receptors. To characterize Netrin signaling, a gain-of-function UNC-40 molecule, MYR::UNC-40 (an UNC-40 fusion protein in which the extracellular and transmembrane domains are deleted and replaced by sequences encoding a membrane-targeting myristoylation signal) is generated. MYR::UNC-40 causes axon guidance defects, excess axon branching, and excessive axon and cell body outgrowth. These defects are suppressed by loss-of-function mutations in ced-10 (a Rac GTPase), unc-34 (an Enabled homolog), and unc-115 (a putative actin binding protein: Drosophila homolog - unc-115). ced-10, unc-34, and unc-115 also function in endogenous unc-40 signaling. These results indicate that Enabled functions in axonal attraction as well as axon repulsion. UNC-40 has two conserved cytoplasmic motifs that mediate distinct downstream pathways: CED-10, UNC-115, and the UNC-40 P2 motif act in one pathway, and UNC-34 and the UNC-40 P1 motif act in the other. Thus, UNC-40 might act as a scaffold to deliver several independent signals to the actin cytoskeleton (Gitai, 2003).

Netrins have been shown to promote outgrowth and guidance: vertebrate Netrin-1 was originally identified based on its ability to enhance axon outgrowth into a collagen matrix, and Netrin-1 knockout mice have defects in axon outgrowth in addition to axon guidance. Netrin can also orient axon outgrowth. Both of these effects of Netrin are dependent on the DCC receptor. The results of this study suggest that MYR::UNC-40 activates cytoplasmic signaling of the UNC-40 pathway in a constitutive, ligand-independent manner. The in vivo activation of signaling by the deletion of the extracellular and transmembrane domains suggests that these domains normally function to prevent UNC-40 activation but are disinhibited when UNC-6 binds to UNC-40. A similar disinhibition model has been proposed for the role of Netrin in activating the DCC-UNC-5 complex for axon repulsion (Gitai, 2003).

Double and triple mutant analysis indicates that all of the myr::unc-40 suppressors, unc-34, ced-10, and unc-115 are likely to participate in the endogenous unc-40 signaling pathway. These results suggest that myr::unc-40 activates the endogenous unc-40 signaling pathway, consistent with its acting as a constitutively active form of unc-40. unc-34, ced-10, and unc-115 were found to signal downstream of unc-40 in two parallel, partially redundant pathways. unc-34/Enabled also plays a partially redundant role in the sax-3/Robo pathway. The activation of parallel signaling modules with some functional overlap or redundancy may be a general feature of axon guidance signaling. It is worth noting that this apparent genetic redundancy could result from disrupting cell biological processes that are actually distinct. The activation of multiple pathways for cytoskeletal remodeling by guidance receptors may contribute to accurate guidance through various physical environments (Gitai, 2003).

MYR::UNC-40 is capable of inducing axon outgrowth, misguidance, branching, and cell body deformation. All of these phenotypes can be suppressed by unc-34, ced-10, and unc-115 or by deletions in the P1 and P2 motifs. These results suggest that distinct effects on cell morphology can be induced by the same signaling pathways, consistent with the observation that Netrin can signal through DCC to regulate cell migration, axon outgrowth, axon attraction, and axon repulsion (Gitai, 2003).

MYR::UNC-40 activity generates new outgrowths even in the adult stage, well past the normal period of neuronal development. It thus seems likely that downstream effectors of UNC-40 persist and remain functional into adulthood. Indeed, reporter gene fusions to unc-115 and ced-10 are expressed throughout the life of C. elegans. One possibility is that these genes function later in development to increase the size of the neuron as the size of the animal increases (Gitai, 2003).

Enabled was initially identified as a dosage-sensitive suppressor of Abl tyrosine kinase mutations in Drosophila. Enabled and its family members UNC-34, Mena, VASP, and EVL share a conserved domain structure that includes an N-terminal EVH1 domain and a C-terminal EVH2 domain. The EVH1 domain binds to proteins containing a FPPPP consensus sequence, found in actin-associated molecules such as zyxin and vinculin, whereas the EVH2 domain has been implicated in oligomerization as well as G and F actin binding (Gitai, 2003).

Enabled proteins can nucleate actin polymerization in vitro. In vivo, Ena proteins are important for a number of actin-based cellular processes including axon guidance, platelet shape change, and Jurkat T cell polarization. Ena proteins were initially thought to promote cellular outgrowth, since VASP enhances the actin-based motility of the intracellular pathogen Listeria monoctytogenes, and overexpression of Mena in fibroblasts produces actin-based outgrowths. However, this view was reversed when enrichment of Mena at the leading edge of fibroblasts was found to decrease motility, while depletion of Mena from the leading edge enhanced motility. These observations led to the idea that Ena proteins negatively affect outgrowth. This idea was reinforced when in Drosophila and C. elegans, UNC-34/Enabled was found to interact physically and genetically with the SAX-3/Robo guidance receptor to mediate axon repulsion. Furthermore, unc-34 mutants suppress the axon repulsion induced by ectopic expression of unc-5, suggesting a role for UNC-34 in mediating repulsion from UNC-6/Netrin. These results created a paradox between the observed role for Enabled in promoting actin-based activities generally associated with stimulation of outgrowth in vitro and its clear roles in axon repulsion and inhibition of cell motility in vivo (Gitai, 2003).

A recent paper provided a potential resolution for this paradox by examining the mechanism by which Mena inhibits fibroblast motility. Mena enrichment at the leading edge was actually found to enhance the dynamics of lamellipodial protrusion; the paradoxical decreased net motility results from the fact that these additional protrusions are not stabilized. These observations led to a proposal that Ena proteins function to stimulate the dynamics of protrusions at the leading edge. Whether the presence of additional protrusions promotes or inhibits cell migration or axon outgrowth may depend on whether the protrusions are stabilized or destabilized. It is thus possible that Mena-induced fibroblast protrusions are not stabilized because the actin filaments within them are isolated and unstable. It is proposed that actin filament bundling, observed in the filopodia of axonal growth cones, could provide a cellular context in which Mena-induced protrusions are stabilized. Thus, this new view of the mechanism of Enabled protein function is potentially consistent with a role for Enabled not just in axon repulsion and outgrowth inhibition, but also in axon attraction (Gitai, 2003).

The results provide direct evidence that UNC-34 can indeed function in an attractive axon guidance pathway: the endogenous UNC-6/UNC-40 pathway. These data establish the idea that Enabled proteins can promote outgrowth and attraction in vivo. In the AVM sensory neuron there is a remarkable example of Enabled's duality, since this single cell uses UNC-34/Ena downstream of both UNC-40 and SAX-3 to promote axon attraction and repulsion, respectively. The mild effect of unc-34 mutations on AVM axon guidance suggests that UNC-34 is not essential for either UNC-40 or SAX-3 function. This finding is consistent with the above model wherein Ena proteins promote outgrowth dynamics but are not dedicated factors required for a specific outgrowth response (Gitai, 2003).

These results identified two distinct pathways that mediate UNC-40 signaling: UNC-34/Enabled acts in one and CED-10/Rac and UNC-115/abLIM act in the other. Rac proteins have previously been shown to play roles in axon guidance, and Rac function is essential for repulsive axon guidance signaling by the Semaphorin receptor, Plexin. The involvement of a Rac protein in Netrin attraction is consistent with the observation that Rac promotes lamellipodial extension, since growth cones have a flattened area with some similarities to lamellipodia. Indeed, recent reports demonstrate that Netrin stimulation can activate Rac in vitro. It is interesting that ced-10 is important in the unc-40 pathway, but both mig-2, which encodes another C. elegans Rac-like protein, and unc-73, which encodes a Guanine Nucleotide Exchange Factor (GEF), are not. In preliminary studies, a mutation in rac-2(ok326), the third Rac-like gene in C. elegans, appears to partially suppress the excess outgrowth of MYR::UNC-40, suggesting that UNC-40 may signal to several, but not all, Rac proteins (Gitai, 2003).

The mechanisms by which Rac proteins cause changes in the actin cytoskeleton during axon guidance are largely unknown. The results suggest that UNC-115 acts as an element in the Rac signaling pathway. The UNC-115 protein contains three LIM domains and a villin headpiece domain. UNC-115 has been proposed to bind actin through its villin headpiece domain; thus, UNC-115 may provide a link between Rac and actin. A different LIM domain-containing protein, LIM-kinase, acts downstream of Rac through a PAK intermediate. The role of UNC-115 in axon guidance is not specific to C. elegans; a dominant-negative form of a vertebrate UNC-115 homolog, abLIM, can cause axon defects in retinal ganglion cells (Gitai, 2003).

Directed-turning toward an axonal attractant requires propagation of spatial information about the source of the attractant to downstream signaling events. Localized signaling might be achieved by localized nucleation of a signaling complex around the activated receptor. The activation of the UNC-34- and CED-10/UNC-115-dependent pathways by UNC-40 correspond to the specific conserved P1 and P2 motifs within the UNC-40 cytoplasmic domain. It is suggested that these actin-regulatory activities may remain closely associated with the activated receptor. UNC-40 may thus function as a scaffold for assembling several independent activities that regulate the cytoskeleton (Gitai, 2003).

During axon guidance, the ventral guidance of the Caenorhabditis elegans anterior ventral microtubule axon is controlled by two cues, the UNC-6/netrin attractant recognized by the UNC-40/DCC receptor and the SLT-1/slit repellent recognized by the SAX-3/robo receptor. Loss-of-function mutations in clr-1 enhance netrin-dependent attraction, suppressing ventral guidance defects in slt-1 mutants. clr-1 encodes a transmembrane receptor protein tyrosine phosphatase (RPTP) that functions in AVM to inhibit signaling through the DCC family receptor UNC-40 and its effector, UNC-34/enabled. The known effects of other RPTPs in axon guidance could result from modulation of guidance receptors like UNC-40/DCC (Chang, 2004).

Integrin acts upstream of netrin signaling to regulate formation of the anchor cell's invasive membrane in C. elegans

Integrin expression and activity have been strongly correlated with developmental and pathological processes involving cell invasion through basement membranes. The role of integrins in mediating these invasions, however, remains unclear. Utilizing the genetically and visually accessible model of anchor cell (AC) invasion in C. elegans, it has been shown that netrin signaling orients a specialized invasive cell membrane domain toward the basement membrane. This study demonstrates that the integrin heterodimer INA-1/PAT-3 plays a crucial role in AC invasion, in part by targeting the netrin receptor UNC-40 (DCC) to the AC's plasma membrane. Analyses of the invasive membrane components phosphatidylinositol 4,5-bisphosphate, the Rac GTPase MIG-2, and F-actin further indicate that INA-1/PAT-3 plays a broad role in promoting the plasma membrane association of these molecules. Taken together, these studies reveal a role for integrin in regulating the plasma membrane targeting and netrin-dependent orientation of a specialized invasive membrane domain (Hagedorn, 2009).

Anchor cell invasion into the vulval epithelium in C. elegans is an in vivo model of invasive behavior that allows for genetic and single-cell visual analysis of invasion. During the mid-L3 larval stage, a basally derived invasive process from the AC crosses the gonadal and ventral epidermal BMs and then moves between the central 1°-fated vulval precursor cells (VPCs) to mediate uterine-vulval attachment. Recent studies have shown that the invasive cell membrane of the AC is a specialized subcellular domain that is polarized toward the BM by the action of the UNC-6 (netrin) pathway. Approximately 4 hr prior to invasion, expression of the secreted guidance cue UNC-6 (netrin) from the ventral nerve cord targets its receptor, UNC-40 (DCC), to the AC's invasive membrane. There, netrin signaling localizes a number of actin regulators that promote invasion, including the Rac GTPases MIG-2 and CED-10, the Ena/VASP ortholog UNC-34, and the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). The proper orientation of these components at the basal membrane is required to generate robust protrusions that breach the BM in response to a later cue from the 1° VPCs that stimulates invasion. Although the molecular components of the invasive membrane are misoriented in unc-6 mutants, they still associate in a nonpolarized manner with the AC's plasma membrane, suggesting that a distinct mechanism exists for regulating their targeting to the cell membrane (Hagedorn, 2009).

Integrins are one of the major cell surface receptors used by metazoan cells to mediate direct cell-matrix interactions. All integrins are heterodimers composed of a single α and β subunit. In vertebrates, integrins have been implicated in regulating cell invasion during blastocyst implantation, angiogenesis, and leukocyte trafficking. Furthermore, the dysregulation of integrin expression and function has been associated with a number of metastatic cancers. Mammals utilize 18 α and 8 β subunits, which combine to form an array of different heterodimers. The complexity of the mammalian integrin receptor family, combined with the difficulty of in vivo analysis has hindered an understanding of the requirement and function of integrin receptors in mediating BM invasion. C. elegans possess only two predicted integrin receptors, composed of an α PAT-2 or α INA-1 subunit bound with the sole β subunit, PAT-3, providing a simplified genetic landscape for examining integrin function (Hagedorn, 2009).

An RNAi screen was conducted to identify additional pathways that regulate invasion, and this study reports that the C. elegans integrin heterodimer INA-1/PAT-3 is a crucial regulator of AC invasion. Cell biological and genetic analyses indicate that INA-1/PAT-3 functions within the AC to control the formation of invasive protrusions that breach the BM. This analysis identifies a key role for integrin in regulating the membrane association of components of the invasive cell membrane, including the netrin receptor UNC-40 (DCC). This work demonstrates an essential role for integrin in controlling BM invasion and reveals an integrin-netrin pathway interaction that mediates the membrane targeting and polarization of the molecular constituents of the AC's invasive membrane (Hagedorn, 2009).

The neogenin/DCC homolog UNC-40 promotes BMP signaling via the RGM protein DRAG-1 in C. elegans

The deleted in colorectal cancer (DCC) homolog neogenin functions in both netrin- and repulsive guidance molecule (RGM)-mediated axon guidance and in bone morphogenetic protein (BMP) signaling. How neogenin functions in mediating BMP signaling is not well understood. This study shows that the sole C. elegans DCC/neogenin homolog UNC-40 positively modulates a BMP-like pathway by functioning in the signal-receiving cells at the ligand/receptor level. This function of UNC-40 is independent of its role in netrin-mediated axon guidance, but requires its association with the repulsive guidance molecule DRAG-1. This study has identified the key residues in the extracellular domain of UNC-40 that are crucial for UNC-40-DRAG-1 interaction and UNC-40 function. Surprisingly, the extracellular domain of UNC-40 is sufficient to promote BMP signaling, in clear contrast to the requirement of its intracellular domain in mediating axon guidance. Mouse neogenin lacking the intracellular domain is also capable of mediating BMP signaling. These findings reveal an unexpected mode of action for neogenin regulation of BMP signaling (Tian, 2013).

Xenopus homologs of Frazzled

A Xenopus DCC homologue (XDCC alpha) is predicted to encode a protein of 1427 amino acids. XDCC alpha and human DCC homologies are greater than 80% identical; each has four immunoglobulin-like domains, six fibronectin type III domains, and a cytoplasmic domain of about 325 amino acids. XDCC alpha expression is present in embryos from stages 19 to 46 in developing forebrain, midbrain, and hindbrain regions. DCC expression is inhibited by treatments that alter the development of mature neural structures: specifically uv-ventralized embryos and exogastrulae with reduced DCC expression. XDCC alpha is expressed as a consequence of neural induction, and unlike some well-characterized tumor suppressor genes, such as the p53 and retinoblastoma genes, that are not differentially expressed in developing Xenopus embryos, the DCC gene may have a specific role in the morphogenesis of the brain and perhaps other tissues and organs (Pierceall, 1994).

Netrin-1 is known to function as a chemoattractant for several classes of developing axons and as a chemorepellent for other classes of axons, apparently dependent on the receptor type expressed by responsive cells. In culture, growth cones of embryonic Xenopus spinal neurons exhibit chemoattractive turning toward the source of netrin-1 but show chemorepulsive responses in the presence of a competitive analog of cAMP or an inhibitor of protein kinase A. Both attractive and repulsive responses are abolished by depleting extracellular calcium and by adding a blocking antibody against the netrin-1 receptor Deleted in Colorectal Cancer. Thus, nerve growth cones may respond to the same guidance cue with opposite turning behavior, dependent on other coincident signals that set the level of cytosolic cAMP (Ming, 1997).

Previous studies have shown that BDNF-induced turning of growth cones also exhibits either attraction or repulsion, depending on differences in cyclic-AMP-dependent activity in neurons. The current studies suggest that Ca2+ signaling (acting downstream from the BDNF receptor known as TrkB) lies upstream from the cAMP-dependent step in the cascade of events, since attractive turning induced by a forskolin gradient is not affected by removal of extracellular CA2+ (Song, 1997). A netrin-induced Ca2+ influx may trigger a rise in cAMP through activation of Ca2+-dependent adenylate cyclases, thus creating a cAMP gradient within the growth cone, a condition known to result in an attractive response of Xenopus growth cones. It is possible that a gradient of cytosolic Ca2+ induced by netrin-1 is responsible for triggering the repulsive response of the growth cone, but the effect is normally overridden by the attractive response due to a cAMP gradient generated by the Ca2+ gradient. Inhibition of cAMP-dependent processes using competitive cAMP analogs may thus unmask the repulsive action of the cytosolic Ca2+ gradient. In principle, cAMP and the cAMP-dependent protein kinase (see Drosophila PKA) pathway could regulate either the receptors for different diffusible guidance cues or the activity of the downstream effector molecules activated by these receptors. The cAMP-dependent protein kinase may thereby act as a gating mechanism, being differentially permissive for a receptor-induced signaling cascade, depending on the cascade's functional status. One group of potential downstream targets of PKA is small GTP-binding proteins of the rho family, e.g., rhoA, rac1 and cdc42, which are known to mediate morphological changes by regulating the actin cytoskeleton and to play a role in growth cone turning. It is known that PKA can phosphorylate rhoA (see Drosophila Rho1), leading to the translocation of membrane-associated rhoA to the cytoplasm and, providing an additional mechanism for its inactivation. The observation that lowering PKA activity converts netrin-1-induced attraction into repulsion suggests the intriguing possibility that activation of an UNC-5-like protein may down-regulate PKA. UNC-5 appears to be either a receptor or a component of a receptor complex, involved in netrin-mediated repulsion. For example, UNC-5 may inhibit adenylate cyclase activity or stimulate phosphodiesterase, which lowers the cAMP level and consequently PKA activity (Ming, 1997).

Netrin-1 promotes outgrowth of axons in vitro through the receptor Deleted in Colorectal Cancer (DCC) and elicits turning of axons within embryonic explants when presented as a point source. It is not known whether netrin-1 alone can elicit turning or whether DCC mediates the turning response. A Xenopus homolog of netrin-1, as in rodents, is expressed in the optic nerve head (disc) and optic nerve; DCC is expressed by RGD axons, consistent with netrin-1 and DCC playing a role in Xenopus RGD axon guidance, similar to that shown in the mouse. Xenopus retinal ganglion cell growth cones orient rapidly toward a pipette ejecting netrin-1, an effect blocked by antibodies to DCC. In vitro, netrin-1 induces a complex growth cone morphology reminiscent of that at the optic nerve head, a site of netrin-1 expression in vivo. These results demonstrate that netrin-1 can function alone to induce turning, implicate DCC in this response, and support the idea that netrin-1 contributes to steering axons out of the retina. The results suggest that netrin-1 might be responsible for the morphological changes observed in RGD growth cones at the optic nerve head in vivo (de la Torre, 1997).

In the developing vertebrate brain, growing axons establish a scaffold of axon tracts connected across the midline via commissures. A population of telencephalic neurons has been discovered that expresses NOC-2, a novel glycoform of the neural cell adhesion molecule N-CAM that is involved in axon guidance in the forebrain. These axons arise from the presumptive telencephalic nucleus, course caudally along the principal longitudinal tract of the forebrain, cross the ventral midline in the midbrain, and then project to the contralateral side of the brain. In the present study mechanisms controlling the growth of these axons across the ventral midline of the midbrain have been investigated. The axon guidance netrin receptor DCC is expressed by the NOC-2 population of axons both within the longitudinal tract and within the ventral midbrain commissure. Mice lacking functional DCC exhibit severe perturbations in the migration of commissural axons toward the floor plate in the spinal cord. In addition, these mice lack several major commissures in the brain, including the corpus callosum and the hippocampal commissure. Disruption of DCC-dependent interactions, both in vitro and in vivo, inhibit the NOC-2 axons from crossing the ventral midbrain. Instead, these axons grew along aberrant trajectories away from the midline, suggesting that DCC-dependent interactions are important for overcoming inhibitory mechanisms within the midbrain of the embryonic vertebrate brain. Thus, coordinated responsiveness of forebrain axons to both chemostimulatory and chemorepulsive cues appears to determine whether they cross the ventral midline in the midbrain (Anderson, 2000

Unc5h, a mammalian homolog of C. elegans Unc5, a UNC-40-interacting protein, is conserved in mammals

Two vertebrate homologs of UNC-5 have been identified that along with UNC-5 and the product of the mouse rostral cerebellar malformation gene (rcm) define a new subfamily of the immunoglobulin superfamily. Their messenger RNAs show prominent expression in various classes of differentiating neurons. UNC5H1 and UNC5H2 are more similar to one another (52% identity) than to UNC-5 (28% identity in each case). Both have two predicted immunoglobulin-like domains and two predicted thrombospondin type-1 repeats in their extracellular domains, a predicted membrane-spanning region, and a large intracellular domain. The cytoplasmic domains do not contain obvious motifs, but do possess a small region of homology to Zona Occludens-1, a protein that localizes to adherens junctions and is implicated in junction formation. ZO-1 contains PDZ domains, structures implicated in protein clustering. Unc5h1 transcripts are detected at the early stages of neural tube development in the ventral spinal cord. At embryonic day 11, when motor neurons begin to differentiate in that region, transcripts are present throughout the ventral spinal cord, excluding the midline floor region, but are most intense in the ventricular zone and at the lateral edges. Unc5h2 transcripts are not detected at significant levels in the spinal cord until E14, when they are found in the roof plate region. These genes are also expressed in non-neural structures. Netrin-1 can bind cells expressing these proteins (Leonardo, 1997).

Mutation of the Unc5h3 (formally known as rcm) gene has important consequences on neuronal migration during cerebellar development. Unc5h3 transcripts are expressed early (embryonic day 8.5) in the hindbrain region and later in the cerebellar primordia. In Unc5h3 mutant embryos, both the development and initial migration of Purkinje cell progenitors occur as in wild-type controls. The rhombic lip, from which granule cell precursors arise, also appears to form normally in mutants. However, at E13.5, an abnormal subpopulation of granule cell and Purkinje cell precursors becomes detectable in rostral areas of the Unc5h3 mutant brain stem. These ectopic cerebellar cells increase in number and continue moving in a rostral direction throughout the remainder of embryogenesis and early stages of postnatal development invading the lateral regions of the pontine area and eventually the inferior colliculus. Cell proliferation markers demonstrate the mitotic nature of these subpial ectopic granule neurons, indicating the displacement of the rostral external germinal layer in mutant animals. These data suggest that establishment of the rostral cerebellar boundary may rely on chemorepulsive signaling events that require UNC5H3 expressed by cerebellar neurons and extracellular ligands that are functionally related to the UNC5H3-binding and guidance molecule, netrin1. Although the phenotype resulting from the Unc5h3 mutation is apparently limited to the formation of the cerebellum, additional sites of Unc5h3 expression are also found during development suggesting the compensatory function of other genes (Przyborski, 1998).

A recently described recessive mouse mutant, rostral cerebellar malformation (rcm/rcm), demonstrates a swaying gait at approximately 12 days of age. The mutant cerebellar (Cb) phenotype consists of cerebellar tissue that extends rostrally, beyond the usual distinct anterior cerebellar boundary, into the midbrain. Interestingly, the cerebellar ectopia occurs in the absence of any significant alterations in the distribution of nuclear groups within the brainstem. The ectopic Cb tissue is (1) adherent to the posterior and lateral aspects of the inferior colliculus and to the lateral aspect of the rostral brainstem and (2) contains acellular regions within the inner granular layer (igl) and ectopic, calbindin-immunoreactive Purkinje cells (PCs) deep relative to the igl. Within the Cb proper, PC organization is generally normal, as revealed by zebrin II immunoreactivity. In the ectopic Cb tissue PCs also exhibit a banded zebrin distribution. Analysis of the spinocerebellar projection in the mutant suggests a lobular distribution similar to that seen in the normal mouse. Within the anterior region, however, the normal parasagittal banding pattern is somewhat obscured. Spinocerebellar innervation of the ectopic Cb tissue exists, but it is almost exclusively confined to the region adjacent to the caudal inferior colliculus. In conjunction with the recent finding that the mutation appears to affect a UNC-5-like receptor protein for netrin-1 (a molecule that may be involved in axonal guidance and cell migration), these results suggest that this mutant is an important model for the analysis of cerebellar development and regionalization (Eisenman, 1998).

Netrins are bifunctional: they attract some axons and repel others. Netrin receptors of the Deleted in Colorectal Cancer (DCC) family are implicated in attraction and those of the UNC5 family in repulsion, but genetic evidence also suggests involvement of the DCC protein UNC-40 in some cases of repulsion. To test whether these proteins form a receptor complex for repulsion, the attractive responses, mediated by DCC, of Xenopus spinal axons to netrin-1 were studied. Attraction is converted to repulsion by expression of UNC5 proteins in these cells. This repulsion requires DCC function; the UNC5 cytoplasmic domain is sufficient to effect the conversion, and repulsion can be initiated by netrin-1 binding to either UNC5 or DCC. The isolated cytoplasmic domains of DCC and UNC5 proteins interact directly, but this interaction is repressed in the context of the full-length proteins. Evidence is presented that netrin-1 triggers the formation of a receptor complex of DCC and UNC5 proteins and simultaneously derepresses the interaction between their cytoplasmic domains, thereby converting DCC-mediated attraction to UNC5/DCC-mediated repulsion (Hong, 1999).

To test whether the ectodomain of UNC5 proteins is required for repulsion, an examination was made of the effect of expressing a chimeric receptor in which the transmembrane and cytoplasmic domains of UNC5H2 (a human UNC5 homolog) were fused to the extracellular domain of DCC. Neurons expressing this DCC/UNC5H2 chimera show the same repulsive response to netrin-1 as do neurons expressing UNC5H2. To determine whether the transmembrane and cytoplasmic domains of UNC5H2 need to be fused to a netrin-binding ectodomain (as is the case for DCC), a chimeric receptor was examined in which the transmembrane and cytoplasmic domains of UNC5H2 were fused to the ectodomain of the NGF receptor TrkA, which does not bind netrin-1. Xenopus spinal neurons do not express TrkA endogenously and do not respond to an NGF gradient with either attraction or repulsion. Neurons expressing the TrkA/UNC5H2 chimera are repelled by netrin-1, a response that is blocked by the anti-DCC antibody; NGF has no effect on these neurons. These results suggested that the cytoplasmic domain of UNC5H2 might be sufficient for repulsion. This possibility was tested by generating a cDNA coding for the cytoplasmic domain of UNC5H2 preceded by a myristoylation sequence that targets cytoplasmic proteins to the inner leaflet of the plasma membrane. Neurons expressing this myristoylated UNC5H2 cytoplasmic domain construct exhibit marked repulsive responses to netrin-1. Thus, expression of the cytoplasmic domain of UNC5H2 is sufficient to convert netrin-mediated attraction to repulsion. It was then shown that netrin-1 triggers the formation of a heterodimeric or heteromultimeric complex involving DCC and UNC5H2 (Hong, 1999).

To further dissect the interaction between DCC and UNC5H2, attempts were made to identify regions in the DCC cytoplasmic domain required for the interaction. The first 46 amino acids are both necessary and sufficient for the interaction. Deletion of the juxtamembrane (JM) region (aas 1120-1148) does not abolish the interaction when performed in the context of the full-length cytoplasmic domain, and conversely, a construct comprising the JM domain alone does not suffice for the interaction. This shows that the JM domain is neither necessary nor sufficient for the interaction and identifies amino acids 1149-1166 as a key stretch required for the interaction. These 18 amino acids comprise the P1 domain, previously identified as a conserved domain among members of the DCC family. However, a construct comprising the P1 domain alone (aas 1149-1466) is not sufficient for the interaction. It is possible that the P1 domain does not fold properly in the absence of some adjacent sequences on either its amino- or carboxy-terminal ends; alternatively, the juxtamembrane region may be redundant with some other region of the cytoplasmic domain, with either one being sufficient but at least one being necessary (Hong, 1999).

Attempts were then made to identify the regions of UNC5 cytoplasmic domains required for DCC binding. Whereas a construct comprising UNC5H2 residues 707-946 is functional, a construct comprising residues 724-946 is not functional. Thus, residues 707-724 are required for binding the DCC cytoplasmic domain. These 18 residues are highly conserved among all previously described UNC5 proteins, and this domain has been termed the DB domain (since it is required for DCC binding). The DB domain is not the only domain required for repulsion, however. Deleting both the C-teminal Death Domain and 113 amino acids between the DB and the DD domains, but leaving the rest intact, including the DB domain, also results in a dominant-negative construct. Thus, sequences between the DD and DB domains are also important for repulsion, as could arise if these sequences are important for binding adaptor proteins. Deletion of the DB domain and all sequences carboxy terminal to it or deletion of all cytoplasmic domain sequences also results in the generation of dominant-negative constructs (Hong, 1999).

A paradox was raised by the finding that the isolated cytoplasmic domains of DCC and UNC5 proteins can interact, yet the full-length proteins do not coprecipitate in the absence of netrin-1. This raises the possibility that the interaction between cytoplasmic domains might be repressed in the context of the full-length proteins. To explore this possibility, a myristoylated cytoplasmic domain of one of the receptors (DCC or UNC5H2) was coexpressed with the full-length version of the other to see if they would coprecipitate. Full-length DCC coprecipitates with the myristoylated UNC5H2 cytoplasmic domain, but only in the presence of netrin-1. Similarly, only a low level of interaction of full-length UNC5H2 with the myristoylated DCC cytoplasmic domain is observed constitutively, and addition of netrin-1 dramatically increases the interaction. These results imply that in the absence of ligand, the UNC5H2 and DCC cytoplasmic domains are largely inaccessible to one another and that addition of netrin-1 causes some change in UNC5H2 and DCC that enables association of their cytoplasmic domains (Hong, 1999).

Why have a mechanism that switches from attraction to repulsion? The answer presumably lies in the fact that growth cones, as they navigate to their targets, change their responsiveness to guidance cues as they progress. Once a growth cone has reached a particular intermediate target, it must change its priorities in order to be able to move on to the next target. For example, commissural axons are initially attracted to the floor plate using netrin-1, but upon crossing the midline, they lose responsiveness to netrin-1. Since the axons continue to express DCC, the switching off must involve some other change. Another switch in growth cone sensitivity at the midline is the acquisition of Slit responsiveness by upregulation of expression of the Robo receptor in Drosophila. Although not yet demonstrated in vivo, it seems likely that there are circumstances where it is desirable not just to switch on or off responsiveness to a particular cue, but rather to convert the responsiveness from attraction to repulsion, to help move the growth cone along. The ability of one receptor to switch responses mediated by another receptor provides an economical means to achieve this end and avoid confusing the growth cone with simultaneous conflicting signals for attraction and repulsion (Hong, 1999 and references).

Mammalian DCC: Homolog of Drosophila Frazzled

Frazzled Evolutionary homologs part 2/2

frazzled: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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