Invertebrate Trk receptors

Dtrk is a Drosophila gene encoding a receptor tyrosine kinase highly related to the trk family of mammalian neurotrophin receptors. The product of the Dtrk gene, gp160Dtrk, is dynamically expressed during Drosophila embryogenesis in several areas of the developing nervous system, including neurons and fasciculating axons. gp160Dtrk has structural homology with neural cell adhesion molecules of the immunoglobulin superfamily and promotes cell adhesion in a homophilic, Ca2+ independent manner. More importantly, this adhesion process specifically activates its tyrosine protein kinase activity. These findings suggest that gp160Dtrk represents a new class of neural cell adhesion molecules that may regulate neuronal recognition and axonal guidance during the development of the Drosophila nervous system. Dtrk, shows limited similarity to vertebrate Trk proteins in its extracellular domain. However, the presence of Ig domains suggests Dtrk activation may be mediated by cell adhesion rather than neurotropic factors (Pulido, 1995).

KIN-8 in C. elegans is highly homologous to human ROR-1 and 2 receptor tyrosine kinases of unknown functions. These kinases belong to a new subfamily related to the Trk subfamily. A kin-8 promoter::gfp fusion gene was expressed in ASI and many other neurons, as well as in pharyngeal and head muscles. A kin-8 deletion mutant has been isolated that shows constitutive dauer larva formation (Daf-c) phenotype: about half of the F1 progeny became dauer larvae when they are cultivated on an old lawn of E. coli as food. Among the cells expressing kin-8::gfp, only ASI sensory neurons are known to express DAF-7 TGF-beta, a key molecule preventing dauer larva formation. In the kin-8 deletion mutant, expression of daf-7::gfp in ASI is greatly reduced; dye-filling in ASI is specifically lost, and ASI sensory processes do not completely extend into the amphid pore. The Daf-c phenotype is suppressed by daf-7 cDNA expression or a daf-3 null mutation. In the kin-8 mutant, ASI-directed expression of kin-8 cDNA under the daf-7 promoter or expression by a heat shock promoter rescues the dye-filling defect, but not the Daf-c phenotype. These results show that the kin-8 mutation causes the Daf-c phenotype through reduction of the daf-7 gene expression, and that KIN-8 function is cell-autonomous for the dye-filling in ASI. KIN-8 is required for the process development of ASI, and also involved in promotion of daf-7 expression through a physiological or developmental function (Koga, 1999).

Alternative models (termed cases A, a, B and b) are considered for KIN-8 function. KIN-8 may be a component in a signal transduction pathway directly controlling daf-7 expression (case A); KIN-8 acts in another pathway that influences or modifies the direct pathway (case a) and the activation of KIN-8 is dynamically regulated by the amount of its ligand, which probably responds to an environmental condition (case B); KIN-8 is constitutively activated (case b). Although no evidence clearly supports any one of these four cases, the B cases (AB or aB) seem interesting. Transcription of the daf-7 gene is controlled by environmental stimuli; the expression is high under non-dauer-inducing conditions (abundant food, a low concentration of dauer pheromone and a low temperature), whereas it is suppressed under dauer-inducing conditions, and these stimuli are sensed by sensory neurons in the amphid. Therefore, in the B cases, a possible ligand for KIN-8 should be secreted under the non-dauer inducing conditions and not under the dauer inducing conditions. Sensory neurons in the amphid may be plausible candidates producing the KIN-8 ligand, because sensing of the environmental stimuli and secretion of a ligand can be simply linked within a single cell and because secretion of an endocrinologic factor is a fairly common property of neurons. This implies a neuroendocrinological signaling pathway in which a sensory neuron perceives environmental information and transforms this information into an amount of the KIN-8 ligand to be secreted. The ligand may be received by KIN-8 in ASI or related cells. The Daf-c phenotype of kin-8 is not temperature-sensitive and similar to that of daf-7;ttx-3 double mutant, which might imply that environmental temperature information could be transduced to ASI through such a KIN-8 pathway (Koga, 1999).

As is KIN-8, the DAF-1/DAF-4 receptor for DAF-7 TGF-beta is expressed in amphid sensory neurons, including ASI and many interneurons. DAF-2 insulin receptor-like molecules may also work in neurons, because a downstream factor, DAF-16 fork head transcription factor, is expressed in the neurons, ectoderm, muscles and intestine. Although a ligand for DAF-2, a presumed insulin-like molecule has not yet been identified: this ligand might also be produced in a sensory neuron. These arguments have led to the speculation that the neurons sensing environmental signals secrete factors such as DAF-7, a DAF-2 ligand and a KIN-8 ligand; then the information represented by these factors is processed and integrated through paracrine, endocrine or autocrine mechanisms among the neurons expressing the corresponding receptors. This type of signaling may be advantageous in a process such as decision between normal development and dauer formation, in which integration of a variety of environmental information over a period of time is probably required. The kin-8 mutant phenotypes in the posterior body part are similar to the phenotypes called withered tails (Wit) in C. elegans that are caused by defects in migration or elongation of the canal-associated-neurons. There may be such defects in the kin-8 mutant. The kin-8 mutant shows some defects in development of ASI sensory process and in migration of DTCs. These results suggest that KIN-8 may be involved in migration of those cells and processes, although specific mechanisms are unknown (Koga, 1999 and references therein).

What are the molecular mechanisms of KIN-8 function? kin-8(ks52) deletes most of the cytoplasmic region, including the kinase domain; it is null for dauer formation and a kinase-negative KIN-8 is functional. Given that little or no KIN-8 truncated protein is present in the kin-8(ks52) mutant, it may be possible that only the extracellular region of KIN-8 is required as a scaffold for other proteins in the wild type. But if kin-8(ks52) is null in spite of the presence of truncated KIN-8, the cytoplasmic region of wild-type KIN-8 is thought to have a function. In this case, a protein is expected to be associated with the KIN-8 cytoplasmic region. This presumed associated protein is possibly activated without the kinase activity of KIN-8 when a ligand binds to KIN-8. A possible mechanism for activation is suggested: Dimerization of KIN-8 upon ligand binding would bring the associated proteins in close contact with one another and would enhance activation. The Unc phenotype of kinase-negative KIN-8 suggests that the kinase activity has another role than dauer formation, if the Unc phenotype represents an authentic KIN-8 function (Koga, 1999).

Ror kinases are members of a family of orphan receptors with tyrosine kinase activity; they are related to muscle specific kinase (MuSK), a receptor tyrosine kinase that assembles acetylcholine receptors at the neuromuscular junction. Although the functions of Ror kinases are unknown, similarities between Ror and MuSK kinases have led to speculation that Ror kinases regulate synaptic development. The C. elegans gene cam-1 encodes a member of the Ror kinase family that guides migrating cells and orients the polarity of asymmetric cell divisions and axon outgrowth. Tyrosine kinase activity is required for some of the functions of CAM-1, but not for its role in cell migration. CAM-1 is expressed in cells that require its function, and acts cell autonomously in migrating neurons. Overexpression and loss of cam-1 function result in reciprocal cell-migration phenotypes, indicating that levels of CAM-1 influence the final positions of migrating cells. These results raise the possibility that Ror kinases regulate cell motility and asymmetric cell division in organisms as diverse as nematodes and mammals (Forrester, 1999).

During Caenorhabditis elegans development, the HSN neurons and the right Q neuroblast and its descendants undergo long-range anteriorly directed migrations. Both of these migrations require EGL-20, a C. elegans Wnt homolog. Through a canonical Wnt signaling pathway, EGL-20/Wnt transcriptionally activates the Hox gene mab-5 in the left Q neuroblast and its descendants, causing the cells to migrate posteriorly. CAM-1, a Ror receptor tyrosine kinase (RTK) family member, inhibits EGL-20 signaling. Excess EGL-20, like loss of cam-1, causes the HSNs to migrate too far anteriorly. Excess CAM-1, like loss of egl-20, shifts the final positions of the HSNs posteriorly and causes the left Q neuroblast descendants to migrate anteriorly. The reversal in the migration of the left Q neuroblast and its descendants results from a failure to express mab-5, an egl-20 mutant phenotype. These data suggest that CAM-1 negatively regulates EGL-20 (Forrester, 2004).

Nicotinic (cholinergic) neurotransmission plays a critical role in the vertebrate nervous system, underlies nicotine addiction, and nicotinic receptor dysfunction leads to neurological disorders. The C. elegans neuromuscular junction (NMJ) shares many characteristics with neuronal synapses, including multiple classes of postsynaptic currents. Two genes required for the major excitatory current found at the C. elegans NMJ have been identified: (1) acr-16 encodes a nicotinic AChR subunit homologous to the vertebrate α7 subunit, and (2) cam-1, which encodes a Ror receptor tyrosine kinase. acr-16 mutants lack fast cholinergic current at the NMJ and exhibit synthetic behavioral deficits with other known AChR mutants. In cam-1 mutants, ACR-16 is mislocalized and ACR-16-dependent currents are disrupted. The postsynaptic deficit in cam-1 mutants is accompanied by alterations in the distribution of cholinergic vesicles and associated synaptic proteins. It is hypothesized that CAM-1 contributes to the localization or stabilization of postsynaptic ACR-16 receptors and presynaptic release sites (Francis, 2005).

Neurotrophins and their Trk receptors play a crucial role in the development and maintenance of the vertebrate nervous system, but to date no component of this signaling system has been found in invertebrates. A molluscan Trk receptor, designated Ltrk, is described from the snail Lymnaea stagnalis. The full-length sequence of Ltrk reveals most of the characteristics typical of Trk receptors, including highly conserved transmembrane and intracellular tyrosine kinase domains, and a typical extracellular domain of leucine-rich motifs flanked by cysteine clusters. Ltrk has a unique N-terminal extension and lacks immunoglobulin-like domains. Ltrk is expressed during development in a stage-specific manner, and also in the adult, where its expression is confined to the central nervous system and its associated endocrine tissues. Ltrk has the highest sequence identity with the TrkC mammalian receptor and, when exogenously expressed in fibroblasts or COS cells, binds human NT-3, but not NGF or BDNF, with an affinity of 2.5 nM. These findings support an early evolutionary origin of the Trk family as neuronal receptor tyrosine kinases and suggest that Trk signaling mechanisms may be highly conserved between vertebrates and invertebrates. Drosophila RTKs (Dtrk, Dnrk and Dror), although Trk-related in their intracellular sequences, have very different extracellular domains, and thus are not considered to be Trk receptors (van Kesteren, 1998).

The C. elegans ROR receptor tyrosine kinase, CAM-1, non-autonomously inhibits the Wnt pathway

Inhibitors of Wnt signaling promote normal development and prevent cancer by restraining when and where the Wnt pathway is activated. ROR proteins, a class of Wnt-binding receptor tyrosine kinases, inhibit Wnt signaling by an unknown mechanism. To clarify how RORs inhibit the Wnt pathway, the relationship between Wnts and the sole C. elegans ROR homolog, cam-1, was examined during C. elegans vulval development, a Wnt-regulated process. It was found that loss and overexpression of cam-1 causes reciprocal defects in Wnt-mediated cell-fate specification. The molecular and genetic analyses revealed that the CAM-1 extracellular domain (ECD) is sufficient to non-autonomously antagonize multiple Wnts, suggesting that the CAM-1/ROR ECD sequesters Wnts. A sequestration model is supported by findings that the CAM-1 ECD binds to several Wnts in vitro. These results demonstrate how ROR proteins help to refine the spatial pattern of Wnt activity in a complex multicellular environment (Green, 2007).

Despite studies in several different organisms, the mechanism of ROR action remains elusive. This work characterized the role of CAM-1/ROR as a regulator of Wnt distribution and determined that one function of ROR proteins is to sequester Wnts. It has been hypothesized that CAM-1/ROR could sequester Wnts. Kim (2003) found that expression of the membrane-anchored CAM-1 ECD was sufficient to rescue the cell migration defects of cam-1(lf) worms and that overexpression of the membrane-anchored CAM-1 CRD caused defects in HSN and Q cell migration similar to those caused by mutation of egl-20/Wnt, leading these authors to propose that the CAM-1 CRD might sequester EGL-20/WNT. Indeed, CAM-1 was later shown to inhibit EGL-20 signaling in cell migration independently of the CAM-1 cytoplasmic domain (Forrester, 2004). However, the mechanism of this inhibition was not demonstrated. In particular, since the ROR2 CRD is capable of dimerizing with Fz (Oishi, 2003), the CAM-1 ECD could potentially function cell-autonomously by inhibiting the Wnt receptor (Green, 2007).

Genetic data indicate that CAM-1 antagonizes Wnt signaling during vulval development. It was found that in lin-17 and lin-18 mutant backgrounds, cam-1 mutations cause an 'overinduced' phenotype owing to elevated levels of Wnt activity. Loss of lin-17 or lin-18 might provide a sensitized background if Wnt receptors LIN-17 and LIN-18, like CAM-1, also affect the extracellular distribution of Wnts. According to this hypothesis, mutation of lin-17 or lin-18 would similarly result in elevated extracellular Wnt levels; however, the data do not conclusively support this hypothesis (Green, 2007).

Using vulval development as a model, this study shows conclusively that CAM-1/ROR can act non-autonomously. The source of the Wnts required for vulval induction is unknown and a sequestration model would require that Pmyo-3::CAM-1::GFP (muscle expression) and Psnb-1::CAM-1::GFP (neuronal expression) are expressed in positions that enable them to restrict diffusion or transport of the Wnts to the VPCs. EGL-20/WNT forms a gradient of decreasing concentration from its site of expression in the tail extending anteriorly past the VPCs (Coudreuse, 2006). The distance between the source of EGL-20 and the VPCs provides ample opportunity for CAM-1 expressed in nervous or muscle tissue to prevent EGL-20 from reaching the VPCs. CWN-1/WNT is expressed in ventral cord neurons (VCNs) and posterior body wall muscle. Endogenous CAM-1 expression in body wall muscle and VCNs, which are in close proximity to the VPCs, could place CAM-1 between the source of cwn-1 expression and the VPCs, allowing CAM-1 to act as a barrier and limit the amount of Wnt signal received by the VPCs. CAM-1 could also function at the Wnt source to limit secretion. Consistent with inhibition by sequestration, CAM-1 overexpression antagonizes Wnt signaling independently of the cytoplasmic domain. Also, phenotypes of cam-1 mutants indicate that the membrane-anchored ECD is sufficient to inhibit Wnt signaling (Green, 2007).

A sequestration model also predicts that CAM-1 specifically binds to those Wnts that it antagonizes. In agreement with genetic data, it was found that the CAM-1 CRD can bind to Wnts CWN-1, EGL-20 and MOM-2 in vitro. The initial experimental design included measuring binding at various concentrations of CRD-AP that would allow calculation of the binding affinity of each receptor-ligand pair. However, preliminary results showed high background binding to untransfected Drosophila S2 cells. The concentration of CRD-AP was chosen at which the greatest difference existed between binding to Nrt-Wnt-expressing and to untransfected cells, and all of the combinations were tested at this concentration. This study never observed a difference greater than 2-fold. Weak binding could be caused by a species barrier, whereby the Drosophila cells do not express a necessary cofactor or do not process Wnts in a manner conducive to high-affinity binding to C. elegans receptors. Although the binding that was detected is not as robust as that observed for Drosophila Wnts and Fzs, it might still be informative (Green, 2007).

Although sequestration through Wnt-CRD binding can account for many functions of CAM-1/ROR, there are examples in which CAM-1 might function by a different mechanism. The membrane-anchored ECD, but not the membrane-anchored CRD alone, has been shown to be sufficient to rescue all cell migration defects of cam-1(lf) worms. In cases where the CRD is not sufficient, ligand binding might require additional CAM-1 ECD(s) - e.g. the kringle or Ig domain - or these might be cases in which CAM-1 functions by a non-sequestration mechanism. Other examples of CAM-1 function that are probably not due to sequestration include cell-autonomous roles in CAN migration and development of the ASI sensory neuron. Also, CAM-1 function in Pn.aap division orientation in males requires CAM-1 kinase activity. Although this study has furthered the understanding of ROR function, the role of the cytoplasmic domains remains elusive. CAM-1 shares 44% identity in the kinase domain to human ROR1 and ROR2 and none of the 21 invariant amino acids is altered. Although ROR proteins have demonstrated kinase activity, the precise function of this activity has not been identified (Green, 2007).

Genetic and biochemical observations that CAM-1 interacts not only with EGL-20, but also with other Wnts, suggest that CAM-1 is an important general regulator of Wnt activity, rather than a specific EGL-20 antagonist. As a system in which neighboring cells reproducibly adopt distinct fates, vulva induction has enabled this study of how CAM-1 affects the precision of Wnt distribution. The subtle effects observed upon cam-1 manipulation suggest that CAM-1 serves to buffer Wnt levels rather than to dramatically affect Wnt localization. Such buffering mechanisms might provide robustness to the Wnt morphogen gradient. The high degree of similarity between CAM-1 and vertebrate ROR proteins, in addition to the ability of ROR proteins to inhibit Wnt signaling in a kinase-independent manner, suggest a conserved function of ROR proteins to fine-tune the spatial profile of Wnt activity and to help create regions of distinct cell fate in complex multicellular organisms (Green, 2007).

Wnt-Ror signaling to SIA and SIB neurons directs anterior axon guidance and nerve ring placement in C. elegans

Wnt signaling through Frizzled proteins guides posterior cells and axons in C. elegans into different spatial domains. This study demonstrates an essential role for Wnt signaling through Ror tyrosine kinase homologs in the most prominent anterior neuropil, the nerve ring. A genetic screen uncovered cwn-2, the C. elegans homolog of Wnt5, as a regulator of nerve ring placement. In cwn-2 mutants, all neuronal structures in and around the nerve ring are shifted to an abnormal anterior position. cwn-2 is required at the time of nerve ring formation; it is expressed by cells posterior of the nerve ring, but its precise site of expression is not critical for its function. In nerve ring development, cwn-2 acts primarily through the Wnt receptor CAM-1 (Ror), together with the Frizzled protein MIG-1, with parallel roles for the Frizzled protein CFZ-2. The identification of CAM-1 as a CWN-2 receptor contrasts with CAM-1 action as a non-receptor in other C. elegans Wnt pathways. Cell-specific rescue of cam-1 and cell ablation experiments reveal a crucial role for the SIA and SIB neurons in positioning the nerve ring, linking Wnt signaling to specific cells that organize the anterior nervous system (Kemmerdell, 2009).

CWN-2 has an essential role in nerve ring placement. The results suggest that CWN-2 is a ligand for the CAM-1 (Ror) receptor in the SIA and SIB neurons, perhaps with MIG-1 (Frizzled) as a co-receptor. In the absence of this signaling pathway, many axons and cell bodies in the nerve ring are displaced towards the anterior. The similar effects of Wnt pathway mutations and genetic ablations suggest that SIA and SIB neurons direct normal nerve ring placement. Additional nerve ring guidance genes that act at least partly parallel to cwn-2, cam-1 and mig-1 are the Frizzled gene cfz-2, the Wnt gene cwn-1, and the Robo gene sax-3 (Kemmerdell, 2009).

cwn-2 is required at a discrete time in development, but the site of cwn-2 expression is relatively unimportant. The rescue of cwn-2 mutants by uniform expression or misexpression echoes the rescue of egl-20 and lin-44 Wnt defects by cDNAs expressed from heat-shock promoters, and suggests that C. elegans Wnts can sometimes function as non-spatial cues. For example, CWN-2 could stimulate axon outgrowth of SIA and SIB at a particular time, with spatial information provided by the distribution of receptors or by other guidance cues near the nerve ring, such as UNC-6 and SLT-1. Alternatively, cwn-2 activity could be spatially limited by cell-specific post-translational pathways or by extracellular Wnt-binding proteins. Finally, additional Wnts, such as CWN-1, might contribute spatial information when CWN-2 is misexpressed: disrupting cwn-2 alone may not eliminate the overall posteriorly biased pattern of Wnt expression. Indeed, in the posterior body, overlapping functions of lin-44, egl-20 and cwn-1 can mask the effects of misexpressing a single Wnt (Kemmerdell, 2009).

CAM-1 has been proposed to act as an extracellular inhibitor of Wnts owing to its non-cell-autonomous action in vulval development and the apparent dispensability of its intracellular domain. However, the CAM-1-related protein Ror2 is an established tyrosine kinase receptor for mammalian Wnts, although kinase-independent functions are also known for vertebrate Rors. Nerve ring development initially appeared not to require the intracellular domain of CAM-1, but many double mutants that included the Frizzleds mig-1, cfz-2 and lin-17 and the LRP-like mig-13 uncovered a requirement for the intracellular domain. Together with a specific requirement for cam-1 expression in the SIA and SIB neurons, these results support a receptor function of CAM-1 in nerve ring development. The overlapping expression and rescue of cam-1 and mig-1 in SIA and SIB matches the genetic results suggesting that they act together in a common process, perhaps as co-receptors for CWN-2. In mammalian osteocytes and lung epithelial cells, Frizzled and Ror or Ryk receptors can function together in a signaling complex. The relevant cellular sites of action for cfz-2, lin-17 and mig-13 are unknown, and expression of cfz-2 in SIA and SIB neurons did not rescue cfz-2 mutants, suggesting that cfz-2 has primary functions outside of SIA and SIB. It is too early to determine whether CFZ-2, LIN-17 and MIG-13 might also be CAM-1 co-receptors (Kemmerdell, 2009).

One interesting implication of the use of multiple Wnt receptors is that spatially and temporally restricted receptor expression might be as important in development as restricted ligand expression. Rather than responding passively to an instructive Wnt cue, developing neurons can shape their response to Wnts through their receptor complement. They can also shape the response of more-distant cells by capturing Wnt ligands, as shown for CAM-1 near the vulva (Kemmerdell, 2009).

Cell-type-specific rescue of cam-1, mig-1 and sax-3 and cell ablation experiments revealed an important role for SIA and SIB neurons in nerve ring placement. Several models could explain cwn-2 effects on SIA and SIB. First, cwn-2 could act in a traditional Wnt patterning role to determine SIA and SIB cell fates; SIA and SIB would then organize nerve ring development through other molecular pathways. However, several SIA and SIB markers are expressed normally in cwn-2 mutants, arguing against a cell fate change (Kemmerdell, 2009).

The model that cwn-2 directly affects axon guidance of SIA and SIB neurons, which in turn instruct the positioning of the nerve ring. SIA and SIB neurons occupy a position near the base of the nerve ring, where they might detect CWN-2, as well as the ventral attractant UNC-6 and the anterior repellent SLT-1. In wild-type animals, the nerve ring axon trajectories of SIA and SIB neurons are unusually complex, consistent with a special patterning role. In cwn-2 and cam-1 mutants, the disruption of axon trajectories in SIA and SIB neurons is more complicated than in other cell types: SIA and SIB have guidance defects at many positions, whereas other neurons simply move to an anterior location. It is suggested that the guidance of SIA and SIB neurons is under the direct control of CWN-2, which generates a temporally precise and spatially less precise signal to form a nerve ring at the correct location. Other nerve ring neurons follow SIA and SIB neurons to this location if possible; if SIA and SIB neurons are misguided or absent, the nerve ring shifts to a more anterior position that might be a default position, or one specified by another guidance cue (Kemmerdell, 2009).

Opposing Wnt pathways orient cell polarity during organogenesis

The orientation of asymmetric cell division contributes to the organization of cells within a tissue or organ. For example, mirror-image symmetry of the C. elegans vulva is achieved by the opposite division orientation of the vulval precursor cells (VPCs) flanking the axis of symmetry. This study characterized the molecular mechanisms contributing to this division pattern. Wnts MOM-2 and LIN-44 are expressed at the axis of symmetry and orient the VPCs toward the center. These Wnts act via Fz/LIN-17 and Ryk/LIN-18, which control beta-catenin localization and activate gene transcription. In addition, VPCs on both sides of the axis of symmetry possess a uniform underlying 'ground' polarity, established by the instructive activity of Wnt/EGL-20. EGL-20 establishes ground polarity via a novel type of signaling involving the Ror receptor tyrosine kinase CAM-1 and the planar cell polarity component Van Gogh/VANG-1. Thus, tissue polarity is determined by the integration of multiple Wnt pathways (Green, 2008).

These results describe the contributions of multiple Wnt pathways to the orientation of cell polarity in the C. elegans vulval epithelium. Because no factor required for the posterior orientation of P5.p or P7.p had previously been identified, this orientation was thought to be signaling independent or 'default'. However, when a new approach was used to reduce Wnt levels in a spatiotemporally controlled manner (overexpression of Ror/CAM-1, a Wnt sink), the VPCs displayed instead a randomized orientation, which is likely to be the true default. The posterior orientation seen in the absence of Fz/lin-17 and Ryk/lin-18 depends on the instructive activity of Wnt/EGL-20. This polarity is referred to as 'ground' polarity. In response to centrally located Wnt/MOM-2 (and possibly Wnt/LIN-44), the receptors Fz/LIN-17 and Ryk/LIN-18 orient P5.p and P7.p toward the center. This reorientation of P7.p, 'refined' polarity, provides the mirror-image symmetry required for a functional organ (Green, 2008).

That P7.p is oriented toward the center in wild-type worms suggests that Wnts LIN-44 and MOM-2 have a greater ability to affect P7.p orientation than does EGL-20. Although the posterior-anterior EGL-20 gradient reaches the VPCs, EGL-20 levels may be much lower here than the levels of Wnts secreted from the nearby AC. Indeed, it was found that local expression of egl-20 in the AC can overcome the effects of distally expressed egl-20. lin-44 is expressed in the tail in addition to the AC but has not been shown to have long-range activity. It is thus possible that this posterior source of lin-44 does not affect P7.p orientation and that LIN-44, in addition to MOM-2, acts as a central cue (Green, 2008).

LIN-17 and LIN-18 were previously reported to reorient P7.p and to reverse the AP pattern of nuclear TCF/POP-1 levels in P7.p daughters. This study extended knowledge of the signaling downstream of Fz/LIN-17 and Ryk/LIN-18 by showing that these receptors control the asymmetric localization of two β-catenins, SYS-1 and BAR-1, the first evidence that Ryk proteins regulate β-catenin. Although asymmetric localization of SYS-1 suggests involvement of the Wnt/β-catenin asymmetry pathway, disruption of pathway components either did not cause a P-Rvl phenotype (lit-1(rf)) or caused only a weakly penetrant P-Rvl phenotype [pop-1(RNAi), sys-1(rf), and wrm-1(rf)], making the function of the Wnt/β-catenin asymmetry pathway in refined polarity unclear. LIN-17 and LIN-18 were also shown to activate transcription in the proximal VPC daughters. Yet, this transcription is not required for P7.p reorientation, since transcriptional states observed by POPTOP, a reporter of Wnt target genes, do not always correspond with the morphological phenotype. Therefore, refined polarity may be largely independent of BAR-1 or the Wnt/β-catenin asymmetry pathway and instead be analagous to the spindle reorientation of the EMS cell during C. elegans embryogenesis, in which Wnt signaling affects the cytoskeleton independent of Wnt's effect on gene expression (Green, 2008).

What then, is the purpose of the Wnt/β-catenin asymmetry pathway in the VPCs? The weakly penetrant A-Rvl phenotype seen in wrm-1(rf) and lin-17(lf); lit-1(lf) worms, combined with the observation that EGL-20 regulates SYS-1 asymmetry, suggests that the Wnt/β-catenin asymmetry pathway functions in ground polarity. Therefore, both ground and refined polarity may converge on regulation of these components, although they are not absolutely required for refined polarity. Because the localization of Wnt/β-catenin asymmetry pathway components in ground polarity matches the reiterative pattern seen in most other asymmetric cell divisions in C. elegans, it is hypothesized that localization of these components is initially established as part of a global anterior-posterior polarity. It is likely that LIN-17 and LIN-18 overcome ground polarity by inhibiting the Wnt/β-catenin asymmetry pathway, a scenario consistent with the ability of lit-1(rf) to suppress lin-17(lf) and lin-18(lf) mutations (Green, 2008).

Remarkably, it is only by peeling back the layer of refined polarity that ground polarity can be observed and manipulated. By doing so, it was found that Wnt/EGL-20, expressed from a distant posterior source, imparts uniform AP polarity to the field of VPCs via a new pathway involving Van Gogh/vang-1, a core PCP pathway component. It is noteworthy that Fz is also a core PCP pathway component, yet it does not seem to be involved in EGL-20 signaling via VANG-1. This is not incompatible with other descriptions of PCP. For example, in the Drosophila wing, Van Gogh and Fz antagonize each other and cause wing hairs to orient in opposite directions. The molecular mechanism by which VANG-1 functions in ground polarity is unknown; however, regulation of SYS-1 by VANG-1 provides evidence that the pathway involving egl-20 and vang-1is associated with the Wnt/β-catenin asymmetry pathway (Green, 2008).

A major difference between VPC orientation in C. elegans and PCP in Drosophila is that no Wnt has been directly implicated in Drosophila PCP. Therefore, VPC orientation may be more similar to some forms of PCP in vertebrates. For example, Wnts are believed to act as permissive polarizing factors during vertebrate convergent extension. Also, VPC orientation is strikingly similar to hair cell orientation in the utricular epithelia of the mammalian inner ear, wherein hair cells flanking the axis of symmetry are oriented in opposite directions. In this system, both medial and lateral hair cells possess a uniform underlying polarity as evidenced by asymmetric localization of Prickle, a core PCP pathway component, to the medial side of cells in both populations. Van Gogh is required for proper Prickle asymmetry, perhaps similarly to the role of vang-1 in ground polarity of the VPCs. It is not understood how the position of the utricular axis of symmetry is determined, but the similarities between these two systems suggest that it may represent a local source of Wnt (Green, 2008).

By moving the source of EGL-20 from the posterior to the anterior side of P7.p and thereby reversing P7.p orientation, this study showed that EGL-20 acts as a directional cue. Although it is not presently clear if the pathway involving egl-20 and vang-1 is mechanistically similar to the PCP pathway described in Drosophila and vertebrates, the result nonetheless provides a long-sought example of a Wnt that acts instructively via a PCP pathway component. Detailed description of the subcellular localization of Van Gogh/VANG-1 and other PCP pathway components in the VPCs will be required to make meaningful comparisons between VPC orientation and established models of PCP (Green, 2008).

In addition to vang-1, a role of Ror/cam-1 in ground polarity was identified. The results provide the first evidence that Ror proteins interpret directional Wnt signals, as well as the first evidence that they interact with Van Gogh. Although a Xenopus Ror homolog, Xror2, was previously described to function in PCP during convergent extension, a recent report indicates that the involvement of Xror2 in convergent extension (CE) is actually via a different pathway. In response to Wnt5a, Xror2 activates JNK by a mechanism requiring Xror2 kinase activity. In contrast to Wnt5a/Xror2 signaling, Ror/CAM-1 function in ground polarity does not require JNK. Therefore, the ground polarity pathway involving Wnt/EGL-20, Ror/CAM-1, and Van Gogh/VANG-1 may be a new type of Wnt signaling (Green, 2008).

Using C. elegans vulva development as a model, this study showed that multiple coexisting Wnt pathways with distinct ligand specificities and signaling mechanisms act in concert to regulate the polarity of individual cells during their assembly into complex structures (Green, 2008).

The conserved transmembrane RING finger protein PLR-1 downregulates Wnt signaling by reducing Frizzled, Ror and Ryk cell-surface levels in C. elegans

Wnts control a wide range of essential developmental processes, including cell fate specification, axon guidance and anteroposterior neuronal polarization. This study identified a conserved transmembrane RING finger protein, PLR-1, that governs the response to Wnts by lowering cell-surface levels of the Frizzled family of Wnt receptors in Caenorhabditis elegans. Loss of PLR-1 activity in the neuron AVG causes its anteroposterior polarity to be symmetric or reversed because signaling by the Wnts CWN-1 and CWN-2 are inappropriately activated, whereas ectopic PLR-1 expression blocks Wnt signaling and target gene expression. Frizzleds are enriched at the cell surface; however, when PLR-1 and Frizzled are co-expressed, Frizzled is not detected at the surface but instead is colocalized with PLR-1 in endosomes. The Frizzled cysteine-rich domain (CRD) and invariant second intracellular loop lysine are crucial for PLR-1 downregulation. The PLR-1 RING finger and protease-associated (PA) domain are essential for activity. In a Frizzled-dependent manner, PLR-1 reduces surface levels of the Wnt receptors CAM-1/Ror and LIN-18/Ryk (see Drosophila Derailed). PLR-1 is a homolog of the mammalian transmembrane E3 ubiquitin ligases RNF43 and ZNRF3, which control Frizzled surface levels in an R-spondin-sensitive manner. It is proposed that PLR-1 downregulates Wnt receptor surface levels via lysine ubiquitylation of Frizzled to coordinate spatial and temporal responses to Wnts during neuronal development (Moffat, 2014).

Robo and Ror function in a common receptor complex to regulate Wnt-mediated neurite outgrowth in Caenorhabditis elegans

Growing axons are exposed to various guidance cues en route to their targets, but the mechanisms that govern the response of growth cones to combinations of signals remain largely elusive. This study found that the sole Robo receptor, SAX-3 (see Drosophila Robo), in Caenorhabditis elegans functions as a coreceptor for Wnt/CWN-2 (Drosophila homolog: Wnt5) molecules. SAX-3 binds to Wnt/CWN-2 and facilitates the membrane recruitment of CWN-2. SAX-3 forms a complex with the Ror/CAM-1 receptor and its downstream effector Dsh/DSH-1, promoting signal transduction from Wnt to Dsh. sax-3 functions in Wnt-responsive cells and the SAX-3 receptor is restricted to the side of the cell from which the neurite is extended. DSH-1 has a similar asymmetric distribution, which is disrupted by sax-3 mutation. Taking these results together, it is proposed that Robo receptor can function as a Wnt coreceptor to regulate Wnt-mediated biological processes in vivo (Wang, 2018).

Vertebrate Ror receptor tyrosine kinases

Human cDNA clones encoding two novel proteins with a region strongly homologous to the tyrosine kinase domain of growth factor receptors, in particular of the Trk family, were obtained by a polymerase chain reaction-based approach. These proteins, Ror1 and Ror2, share 58% overall amino acid identity and a structure indicative of cell surface molecules. A secretion signal sequence and a transmembrane domain delimit the extracellular portion, which contains immunoglobulin-like, cysteine-rich, and kringle domains. The cytoplasmic portion contains the tyrosine kinase-like domain which (in Ror2) appears to be associated with protein kinase activity in vitro, followed by serine/threonine- and proline-rich motifs. Partial nucleotide sequences of the rat genes reveal striking evolutionary conservation of the proteins between human and rat. The level of expression of the rat genes is high in the head and body of early embryo and decreases dramatically after embryonic day 16. Based on these data, Ror1 and Ror2 appear to define a new developmentally regulated family of cell surface receptors for unidentified ligands (Masiakowski, 1992).

Drosophila neurospecific receptor tyrosine kinases (RTKs), Dror and Dnrk, as well as Ror1 and Ror2 RTKs, isolated from human neuroblastoma, have been identified as a structurally related novel family of RTKs (Ror-family RTKs). Thus far, little is known about the expression and function of mammalian Ror-family RTKs. Murine Ror-family RTKs, mRor1 and mRor2, have been identified. Both mRor1 and mRor2 genes are induced upon neuronal differentiation of P19EC cells. During neuronal differentiation in vitro, the expression of mRor2 is transiently induced, although that of mRor1 increases continuously. During embryogenesis, the mRor1 gene is expressed in the developing nervous system within restricted regions and in the developing lens epithelium. The expression of mRor1 is sustained in the nervous system and is also detected in non-neuronal tissues after birth. In contrast, the expression of mRor2 is detected mainly in the developing nervous system within broader regions and declines after birth. Possible relationships of mRor1 and mRor2 genes with previously identified mutants have also been examined. It is concluded that the developmental expressions of mRor1 and mRor2, in particular in the nervous system, are differentially regulated, reflecting their expression patterns in vitro. mRor1 and mRor2 may thus play differential roles during the development of the nervous system (Oishi, 1999).

Receptor tyrosine kinases often have critical roles in particular cell lineages by initiating signaling cascades in those lineages. Examples include the neural-specific TRK receptors, the VEGF and angiopoietin endothelial-specific receptors, and the muscle-specific MUSK receptor. Many lineage-restricted receptor tyrosine kinases were initially identified as 'orphans' homologous to known receptors, and only subsequently used to identify their unknown growth factors. Some receptor-tyrosine-kinase-like orphans still lack identified ligands as well as biological roles. One such orphan is encoded by Ror2. Disruption of mouse Ror2 leads to profound skeletal abnormalities, with essentially all endochondrally derived bones foreshortened or misshapen, albeit to differing degrees. Further, Ror2 is selectively expressed in the chondrocytes of all developing cartilage anlagen, where it is essential during initial growth and patterning, as well as subsequently in the proliferating chondrocytes of mature growth plates, where it is required for normal expansion. Thus, Ror2 encodes a receptor-like tyrosine kinase that is selectively expressed in, and particularly important for, the chondrocyte lineage (DeChira, 2000).

Drosophila neurospecific receptor tyrosine kinases (RTKs), Dror and Dnrk, as well as Ror1 and Ror2 RTKs, isolated from human neuroblastoma, have been identified as a structurally related novel family of RTKs (Ror-family RTKs). Murine Ror-family RTKs, mRor1 and mRor2, have been identified. Both mRor1 and mRor2 genes are induced upon neuronal differentiation of P19EC cells. During neuronal differentiation in vitro, the expression of mRor2 is transiently induced, although that of mRor1 increases continuously. During embryogenesis, the mRor1 gene is expressed in the developing nervous system within restricted regions and in the developing lens epithelium. The expression of mRor1 is sustained in the nervous system and is also detected in non-neuronal tissues after birth. In contrast, the expression of mRor2 is detected mainly in the developing nervous system within broader regions and declines after birth. Possible relationships of mRor1 and mRor2 genes with previously identified mutants have also been examined. Thus, the developmental expressions of mRor1 and mRor2, in particular in the nervous system, are differentially regulated, reflecting their expression patterns in vitro. mRor1 and mRor2 may thus play differential roles during the development of the nervous system (Oishi, 2001).

The extracellular regions of mRor1 and mRor2, as well as hRor1 and hRor2 can be divided into several domains, i.e., an Ig-like domain, a cys-rich domain, and a membrane-proximal kringle domain, which are characteristic features of the Ror-family RTKs, although Dnrk and Dror lack the N-terminal Ig-like domain. Among all known RTKs, only the Ror-family RTKs and the Torpedo RTK possess the kringle domain. The kringle domain is a highly folded structure, rich in cysteines, and is found in blood coagulation proteins, apolipoprotein(a), and hepatocyte growth factors. Since it has been envisaged that the kringle domains are involved in mediating protein-protein interaction, these domains in the Ror-family RTKs may be required for associating with their putative ligands. Interestingly, the extracellular regions of the Ror-family RTKs also display some degree of similarity with those of muscle-specific RTKs, the Torpedo RTK and the mammalian MuSKs (Oishi, 2001).

Like hRor1 and hRor2 and the Drosophila Ror-family RTKs, Dnrk and Dror, the cytoplasmic regions of mRor1 and mRor2 contain TK domains that are most similar to those of the Trk-family RTKs. Conserved tyrosine residues (cis regulatory phosphorylation sites) among the Trk-family RTKs, that are involved in the regulation of Trk kinase activity and transphosphorylation, are also found in members of the Ror-family RTKs. Although both Dnrk and Dror possess the canonical ATP binding motifs (GXGXXG/K) within their TK domains, mammalian Ror-family RTKs bear the unusual amino acid substitutions within their putative ATP binding motifs. Interestingly, mammalian, but not Drosophila Ror-family RTKs, possess domains that are rich in prolines (the proline-rich domains) at their cytoplasmic C-terminal portion, that may interact with the Src homology 3 (SH3) domains of cellular adaptor/signaling molecules. In addition, both mRor1 and hRor1, but not mRor2 or hRor2, possess the consensus motif XPPXY within their proline-rich domains, that can bind to the WW domain proteins implicated in several human diseases. It has been speculated that the XPPXY motif can bind to the Src homology 2 (SH2) domain of cellular proteins upon phosphorylation of the tyrosine residue within the motif. Furthermore, mRor2, hRor2 and Dnrk, but not mRor1, hRor1 and Dror, possess a tyrosine-containing motif YALM (Y722ALM725 in the case of mRor2 and hRor2), that can interact with SH2 domains of Shc, Csk and the p85 subunit of phosphoinositide 3'-kinase (PI3K) upon tyrosine phosphorylation. It is important to note that the tyrosine residue, found in this YALM motif, is one of the cis regulatory phosphorylation sites conserved among the Trk- and Ror-family RTKs (Oishi, 2001).

The frizzled (FRZ) module is a novel module type that was first identified in G-protein-coupled receptors of the frizzled and smoothened families and has since been shown to be present in several secreted frizzled-related proteins, in some modular proteases, in collagen XVIII, and in various receptor tyrosine kinases of the Ror family. The FRZ modules constitute the extracellular ligand-binding region of frizzled receptors and are known to mediate signals of WNT family members through these receptors. With an eye toward defining the structure of this important module family, the FRZ domain of rat Ror1 receptor tyrosine kinase was expressed in Pichia pastoris. By proteolytic digestion and amino acid sequencing the disulfide bonds were found to connect the 10 conserved cysteines in a 1-5, 2-4, 3-8, 6-10, and 7-9 pattern. Circular dichroism and differential scanning calorimetry studies on the recombinant protein indicate that the disulfide-bonded FRZ module corresponds to a single, compact, and remarkably stable folding domain possessing both alpha-helices and beta-strands (Roszmusz, 2001).

Ror1 and Ror2 are orphan receptor tyrosine kinases that are most closely related to MuSK and the Trk family of neurotrophin receptors. Expressions of murine Ror1 and Ror2 differ markedly at early stages (E8.5-E9.5). At these times, Ror2 is expressed much more widely than Ror1, expression of which is largely restricted to head mesenchyme. At later stages of development (E12.5-E14.5), Ror1 expression expands and Ror2 expression becomes more restricted than at earlier times, although expression of Ror1 continues to be more restricted than that of Ror2. These changes result in overlapping expression domains but with major differences remaining. In many cases Ror1 is expressed in a sub-set of Ror2-expressing tissues; in others, there is complementary expression of Ror1 and Ror2. Ror1 and Ror2 are both expressed in derivatives of all three germ layers and in most organ systems, including the nervous, circulatory, respiratory, digestive, urogenital and skeletal systems. Conspicuous themes are the expression in major sense organs, and in neural crest and its derivatives (Al-Shawi, 2001).

Spemann organizer plays a central role in neural induction, patterning of the neuroectoderm and mesoderm, and morphogenetic movements during early embryogenesis. By seeking genes whose expression is activated by the organizer-specific LIM homeobox gene Xlim-1 in Xenopus animal caps, the receptor tyrosine kinase Xror2 was isolated. Xror2 is expressed initially in the dorsal marginal zone, then in the notochord and the neuroectoderm posterior to the midbrain-hindbrain boundary. mRNA injection experiments revealed that overexpression of Xror2 inhibits convergent extension of the dorsal mesoderm and neuroectoderm in whole embryos, as well as the elongation of animal caps treated with activin, whereas it does not appear to affect cell differentiation of neural tissue and notochord. Interestingly, mutant constructs in which the kinase domain was point-mutated or deleted (named Xror2-TM) also inhibited convergent extension, and did not counteract the wild-type, suggesting that the ectodomain of Xror2 per se has activities that may be modulated by the intracellular domain. In relation to Wnt signaling for planar cell polarity, the following is observed: (1) the Frizzled-like domain in the ectodomain is required for the activity of wild-type Xror2 and Xror2-TM; (2) co-expression of Xror2 with Xwnt11, Xfz7, or both, synergistically inhibits convergent extension in embryos; (3) inhibition of elongation by Xror2 in activin-treated animal caps is reversed by co-expression of a dominant negative form of Cdc42 that has been suggested to mediate the planar cell polarity pathway of Wnt, and (4) the ectodomain of Xror2 interacts with Xwnts in co-immunoprecipitation experiments. These results suggest that Xror2 cooperates with Wnts to regulate convergent extension of the axial mesoderm and neuroectoderm by modulating the planar cell polarity pathway of Wnt (Hikasa, 2005).

Wnt-5A/Ror2 regulate expression of XPAPC through an alternative noncanonical signaling pathway

XWnt-5A, a member of the nontransforming Wnt-5A class of Wnt ligands, is required for convergent extension (CE) movements in Xenopus embryos. XWnt-5A knockdown phenocopies paraxial protocadherin (XPAPC) loss of function: involuted mesodermal cells fail to align mediolaterally, which results in aberrant movements and a selective inhibition of constriction. XWnt-5A depletion was rescued by coinjection of XPAPC RNA, indicating that XWnt-5A acts upstream of XPAPC. XWnt-5A, but not XWnt-11, stimulates XPAPC expression independent of the canonical Wnt/β-catenin pathway. Transcriptional regulation of XPAPC by XWnt-5A requires the receptor tyrosine kinase Ror2. XWnt-5A/Xror2 signal through PI3 kinase and cdc42 to activate the JNK signaling cascade with the transcription factors ATF2 and c-jun. The Wnt-5A/Ror2 pathway represents an alternative, distinct branch of noncanonical Wnt signaling that controls gene expression and is required in the regulation of convergent extension movements in Xenopus gastrulation (Sachambony, 2007).

The Wnt-5A/Ror2 pathway has to be considered a noncanonical Wnt pathway, because it does not involve β-catenin and LEF/TCF transcription factors. Like other noncanonical Wnt pathways, both gain of function and loss of function inhibited CE movements. However, while XWnt-5A depletion selectively affected constriction in Keller open-face explants, coinjection of high amounts of an MO-insensitive XWnt-5A RNA caused a strong inhibition of Keller open-face explant elongation. This phenotype had been reported earlier for XWnt-5A overexpression, and similarly for Xror2 overexpression, and is most likely due to the inhibition of canonical Wnt signaling. The results further exclude a PTX-sensitive G protein, PKC, CamK II, and NF-AT as downstream effectors of Wnt-5a/Ror2 signaling, which is in agreement with the observation that Wnt-5a/Ror2 antagonized canonical Wnt signaling in cell culture independent of PTX and without modulation of intracellular Ca2+ levels. Thus, Ror2-mediated Wnt-5A signaling is clearly not related to the Wnt/Ca2+ pathway that has so far been associated with XWnt-5A in early Xenopus development (Sachambony, 2007).

The second, well-characterized branch of noncanonical Wnt signaling, the Wnt/PCP pathway, is mediated by Frizzled, Dishevelled, Daam 1, and the small GTPases Rho A and Rac 1. Dsh is a multidomain protein involved in canonical and PCP signaling. It has been shown that canonical signaling requires the DIX and PDZ domains, while PCP signaling utilizes the PDZ and DEP domains of Dsh. Deletion mutants that lack specific domains are used to discriminate between canonical and noncanonical activity of Dsh. A mutant lacking the DIX domain has been shown to activate PCP signaling and to rescue the phenotypes induced by a dominant negative Wnt-11 mutant in Xenopus. Consistent with the assumption that XWnt-11 activates the Wnt/PCP pathway, XWnt-11 depletion was rescued by coinjection of DshΔDIX, but XWnt-5A MO was not. Additionally, XWnt-5A loss of function was not rescued by constitutively active Rac 1 and was only partially rescued by constitutively active Rho A. The latter is probably due to the role of Rho A downstream of XPAPC. Together with the observation that XWnt-11 was not able to replace XWnt-5A and had no influence on XPAPC transcription, these results demonstrate that XWnt-5A and XWnt-11 are not redundant, and that XWnt-5A/Xror2 signaling is not related to the Wnt/PCP pathway. It is concluded that Wnt-5A/Ror2 signaling should be considered as an additional, distinct branch of noncanonical Wnt signaling (Sachambony, 2007).

Canonical and noncanonical Wnts use a common mechanism to activate completely unrelated coreceptors

Wnt ligands signal through β-catenin and are critically involved in cell fate determination and stem/progenitor self-renewal. Wnts also signal through β-catenin-independent or noncanonical pathways that regulate crucial events during embryonic development. The mechanism of noncanonical receptor activation and how Wnts trigger canonical as opposed to noncanonical signaling have yet to be elucidated. This study demonstrates that prototype canonical Wnt3a and noncanonical Wnt5a ligands specifically trigger completely unrelated endogenous coreceptors-LRP5/6 and Ror1/2, respectively-through a common mechanism that involves their Wnt-dependent coupling to the Frizzled (Fzd) coreceptor and recruitment of shared components, including dishevelled (Dvl), axin, and glycogen synthase kinase 3 (GSK3). Ror2 Ser 864 was identified as a critical residue phosphorylated by GSK3 and required for noncanonical receptor activation by Wnt5a, analogous to the priming phosphorylation of low-density receptor-related protein 6 (LRP6) in response to Wnt3a. Furthermore, this mechanism is independent of Ror2 receptor Tyr kinase functions. Consistent with this model of Wnt receptor activation, evidence is provided that canonical and noncanonical Wnts exert reciprocal pathway inhibition at the cell surface by competition for Fzd binding. Thus, different Wnts, through their specific coupling and phosphorylation of unrelated coreceptors, activate completely distinct signaling pathways (Grumolato, 2010).

Neural crest specification by noncanonical Wnt signaling and PAR-1

Neural crest (NC) cells are multipotent progenitors that form at the neural plate border, undergo epithelial-mesenchymal transition and migrate to diverse locations in vertebrate embryos to give rise to many cell types. Multiple signaling factors, including Wnt proteins, operate during early embryonic development to induce the NC cell fate. Whereas the requirement for the Wnt/β-catenin pathway in NC specification has been well established, a similar role for Wnt proteins that do not stabilize β-catenin has remained unclear. Gain- and loss-of-function experiments implicate Wnt11-like proteins in NC specification in Xenopus embryos. In support of this conclusion, modulation of β-catenin-independent signaling through Dishevelled and Ror2 causes predictable changes in premigratory NC. Morpholino-mediated depletion experiments suggest that Wnt11R, a Wnt protein that is expressed in neuroectoderm adjacent to the NC territory, is required for NC formation. Wnt11-like signals might specify NC by altering the localization and activity of the serine/threonine polarity kinase PAR-1 (also known as microtubule-associated regulatory kinase or MARK), which itself plays an essential role in NC formation. Consistent with this model, PAR-1 RNA rescues NC markers in embryos in which noncanonical Wnt signaling has been blocked. These experiments identify novel roles for Wnt11R and PAR-1 in NC specification and reveal an unexpected connection between morphogenesis and cell fate (Ossipava, 2011).

Noncanonical Wnt ligands, such as Wnt5a and Wnt11, do not stabilizeα-catenin or activate TCF-dependent transcription, but regulate morphogenetic processes that involve changes n cell shape and motility, which are sometimes referred to as planar cell polarity (PCP). The signaling from Wnt5 or Wnt11 is thought to involve Ror and Ryk receptors, small Rho GTPases, Rho-associated kinase, c-Jun N-terminal kinases and intracellular calcium. Although noncanonical Wnt pathways have been shown to function in NC cell migration, their importance for NC specification has remained unclear (Ossipava, 2011).

Craniofacial defects in Wnt5a knockout mice, and in wnt11 (silberblick) and wnt5 (pipetail) zebrafish mutant embryos suggest possible roles for noncanonical Wnt signaling in NC development. The results of this study support the view that noncanonical signaling from Wnt11R is essential for NC specification in Xenopus embryos and that it might act by changing the localization and activity of the polarity kinase PAR-1 (Ossipava, 2011).

PAR proteins are conserved regulators of cell polarity that interact with several embryonic signaling pathways, including the Wnt pathway. PAR-1 associates with Dishevelled (Dvl, or Dsh) and participates in Frizzled-dependent Dvl recruitment. This study shows that PAR-1 is itself required for NC specification and can rescue NC defects in embryos with inhibited Wnt5 and Wnt11 signaling. These findings identify PAR-1 as a molecular target for noncanonical Wnt signaling and reveal an unexpected causal connection between cell polarization and the NC cell fate (Ossipava, 2011).

Mutation of mammalian Ror family members

The autosomal recessive form of Robinow syndrome is a severe skeletal dysplasia with generalized limb bone shortening, segmental defects of the spine, brachydactyly and a dysmorphic facial appearance. The gene mutated in RRS has been mapped to chromosome 9q22, a region that overlaps the locus for autosomal dominant brachydactyly type B. The recent identification of ROR2, encoding an orphan receptor tyrosine kinase, as the gene mutated in brachydactyly type B (BDB1) and the mesomelic dwarfing in mice homozygous for a lacZ and/or a neo insertion into Ror2, make this gene a candidate for RRS. Homozygous missense mutations in both intracellular and extracellular domains of ROR2 are reported in affected individuals from 3 unrelated consanguineous families, and a nonsense mutation that removes the tyrosine kinase domain and all subsequent 3' regions of the gene in 14 patients from 7 families from Oman. The nature of these mutations suggests that RRS is caused by loss of ROR2 activity. The identification of mutations in three distinct domains (containing Frizzled-like, kringle and tyrosine kinase motifs) indicates that these are all essential for ROR2 function (Afzal, 2000).

Inherited limb malformations provide a valuable resource for the identification of genes involved in limb development. Brachydactyly type B (BDB), an autosomal dominant disorder, is the most severe of the brachydactylies and characterized by terminal deficiency of the fingers and toes. In the typical form of BDB, the thumbs and big toes are spared, sometimes with broadening or partial duplication. The BDB1 locus has been mapped to chromosome 9q22 within an interval of 7.5 cM. Mutations in ROR2 are described in three unrelated families with BDB1. Distinct heterozygous mutations (2 nonsense, 1 frameshift) were identified within a 7-amino-acid segment of the 943-amino-acid protein, all of which predict truncation of the intracellular portion of the protein immediately after the tyrosine kinase domain. The localized nature of these mutations suggests that they confer a specific gain of function. Further evidence was obtained for this by demonstrating that two patients heterozygous for 9q22 deletions including ROR2 do not exhibit BDB. Expression of the mouse ortholog, Ror2, early in limb development indicates that BDB arises as a primary defect of skeletal patterning (Oldridge, 2000).

In mammals and Drosophila, the Ror-family receptor tyrosine kinases consist of two structurally related proteins, Ror1 and Ror2, characterized by the extracellular Frizzled-like cysteine-rich domain and membrane proximal kringle domains. Mice lacking Ror2 expression exhibit dwarfism, short limbs and tail, facial anomalies, cardiac septal defects, severe cyanosis and respiratory dysfunction, resulting in neonatal lethality. Furthermore, it has recently been reported that mutations of the Ror2 gene cause Robinow syndrome or Brachydactyly type B in human, indicating that Ror2 plays an essential role in morphogenetic and developmental processes. As an attempt to gain insights into their roles in mouse development, expression patterns of Ror1 and Ror2 during early embryogenesis were examined and compared. Interestingly, at early stages, Ror1 and Ror2 exhibit similar expression patterns in the developing face, including the frontonasal process and pharyngeal arches, which are both derived from cephalic neural crest cells. However, Ror1 and Ror2 exhibit different expression patterns in the developing limbs and brain, where the expression of Ror2 was detected broadly compared with that of Ror1. At a later stage, both genes are expressed in a similar fashion in the developing heart and lung, yet in a distinct manner in the brain and eye (Matsuda, 2001).

Robinow syndrome is a short-limbed dwarfism characterized by abnormal morphogenesis of the face and external genitalia, and vertebral segmentation. The recessive form of Robinow syndrome, particularly frequent in Turkey, has a high incidence of abnormalities of the vertebral column such as hemivertebrae and rib fusions, which is not seen in the dominant form. Some patients have cardiac malformations or facial clefting. A gene for RRS has been mapped to 9q21-q23 in 11 families. Haplotype sharing was observed between three families from Turkey, which localized the gene to a 4.9-cM interval. The gene ROR2, which encodes an orphan membrane-bound tyrosine kinase, maps to this region. Heterozygous (presumed gain of function) mutations in ROR2 were previously shown to cause dominant brachydactyly type B. In contrast, Ror2-/- mice have a short-limbed phenotype that is more reminiscent of the mesomelic shortening observed in RRS. Several homozygous ROR2 mutations were detected that are located upstream from those previously found in BDB. The ROR2 mutations present in RRS result in premature stop codons and predict nonfunctional proteins (van Bokhoven, 2000).

A mouse receptor tyrosine kinase (RTK), mRor2, which belongs to the Ror-family of RTKs consisting of at least two structurally related members, is primarily expressed in the heart and nervous system during mouse development. To elucidate the function of mRor2, mice with a mutated mRor2 locus were generated. Mice with a homozygous mutation in mRor2 die just after birth, exhibit dwarfism, severe cyanosis, and short limbs and tails. Whole-mount in situ hybridization analysis has shown that mRor2 is expressed in the branchial arches, heart and limb/tailbuds, in addition to the developing nervous system. The mutants have cardiac septal defects, mainly a ventricular septal defect. In addition, an examination of the skeletal systems reveals that the mutants have shorter limbs, vertebrae and facial structure, with a particular defect in their distal portions, and that almost no calcification was observed in their distal limbs. Histological examination showsabnormalities in the chondrocytes. These findings suggest that mRor2 plays essential roles in the development of the heart and in limb/tail formation, in particular cardiac septal formation and ossification of distal portions of limbs and tails (Takeuchi, 2000).

The mammalian Ror family of receptor tyrosine kinases consists of two structurally related proteins, Ror1 and Ror2. mRor2-deficient mice exhibit widespread skeletal abnormalities, ventricular septal defects in the heart, and respiratory dysfunction, leading to neonatal lethality. mRor1-deficient mice have no apparent skeletal or cardiac abnormalities, yet they also die soon after birth due to respiratory dysfunction. Interestingly, mRor1/mRor2 double mutant mice show markedly enhanced skeletal abnormalities compared with mRor2 mutant mice. Furthermore, double mutant mice also exhibit defects not observed in mRor2 mutant mice, including a sternal defect, dysplasia of the symphysis of the pubic bone, and complete transposition of the great arteries. These results indicate that mRor1 and mRor2 interact genetically in skeletal and cardiac development (Nomi, 2001).

Robinow syndrome (RS) is a human dwarfism syndrome characterized by mesomelic limb shortening, vertebral and craniofacial malformations and small external genitals. Ror2-/- mice were analyzed as a model for the developmental pathology of RS. The results demonstrate that vertebral malformations in Ror2-/- mice are due to reductions in the presomitic mesoderm and defects in somitogenesis. Mesomelic limb shortening in Ror2-/- mice is a consequence of perturbed chondrocyte differentiation. Moreover, the craniofacial phenotype is caused by a midline outgrowth defect. Ror2 expression in the genital tubercle and its reduced size in Ror2-/- mice makes it likely that Ror2 is involved in genital development. In conclusion, these findings suggest that Ror2 is essential at multiple sites during development. The Ror2-/- mouse provides a suitable model that may help to explain many of the underlying developmental malformations in individuals with Robinow syndrome (Schwabe, 2004).

Neurite elongation and branching are key cellular events during brain development, since they underlie the formation of a properly wired neuronal network. The receptor tyrosine kinases Ror1 and Ror2 modulate the growth of neurites as well as their branching pattern in hippocampal neurons. Upon Ror1 or Ror2 suppression using antisense oligonucleotides or RNA interference (RNAi), neurons extended shorter and less branched minor processes when compared to those in control cells. In addition, Ror-depleted cells elongated longer, albeit less branched, axons than seen in control cells. Conversely, Ror overexpression both in non-neuronal cells and in hippocampal neurons resulted in the enhanced extension of short and highly branched processes. These phenotypes were accompanied by changes in the microtubule-associated proteins MAP1B and MAP2. Taken together, these results support a novel role for Ror receptors as modulators of neurite extension in central neurons (Paganoni, 2005).

Interaction of Trk receptors with ligands and receptor interaction between Trk receptors

The two IgG domains of trkA are essential for NGF binding. The requirement for the two IgG domains was further confirmed by Scatchard analysis and affinity crosslinking with 125I-NGF. These results indicate that NGF binding is crucially dependent upon interactions with the IgG domains of the trkA receptor (Perez, 1995).

The potential for the activation of one Trk receptor by ligand binding to another Trk receptor was explored by determining if transphosphorylation on tyrosine residues can occur between receptors. For most of these experiments, functional chimeric receptors were used that contained the extracellular domain of the human type 2 tumor necrosis factor receptor and the transmembrane and cytoplasmic domains of rat TrkA, TrkB, or TrkC and that, when activated by the tumor necrosis factor, mediated the nerve growth factor-like biological activities in PC12 cells. Despite the presence of different extracellular regions, intermolecular transphosphorylation of homologous cytoplasmic domains occurs between TrkA or TrkB and their cognate chimeras. Heterologous transphosphorylation between TrkB and TrkC kinase domains is also observed when one partner is a chimeric receptor; however, TrkA does not transphosphorylate the TrkB or TrkC kinase domains of chimeric receptors or act as a transphosphorylation substrate for these two receptors. The failure of TrkA to take part in transphosphorylation reactions with TrkB and TrkC was confirmed using the natural receptors. Trk receptor transphosphorylation occurs in the two non-neuronal cell types, but TrkA is excluded from these reactions (Canossa, 1996).

Ror2 modulates the canonical Wnt signaling in lung epithelial cells through cooperation with Fzd2

Wnt signaling is mediated through (1) the beta-catenin dependent canonical pathway and, (2) the beta-catenin independent pathways. Multiple receptors, including Fzds, Lrps, Ror2 and Ryk, are involved in Wnt signaling. Ror2 is a single-span transmembrane receptor-tyrosine kinase (RTK). The functions of Ror2 in mediating the non-canonical Wnt signaling have been well established. The role of Ror2 in canonical Wnt signaling is not fully understood. This study reports that Ror2 also positively modulates Wnt3a-activated canonical signaling in a lung carcinoma, H441 cell line. This activity of Ror2 is dependent on cooperative interactions with Fzd2 but not Fzd7. In addition, Ror2-mediated enhancement of canonical signaling requires the extracellular CRD, but not the intracellular PRD domain of Ror2. Evidence that the positive effect of Ror2 on canonical Wnt signaling is inhibited by Dkk1 and Krm1 suggesting that Ror2 enhances an Lrp-dependent STF response. The current study demonstrates the function of Ror2 in modulating canonical Wnt signaling. These findings support a functional scheme whereby regulation of Wnt signaling is achieved by cooperative functions of multiple mediators (Li, 2008; full text of article).

Signaling downstream of Trk receptors

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) selectively bind to distinct members of the Trk family of tyrosine kinase receptors, but all three bind with similar affinities to the neurotrophin receptor p75 (p75NTR). The biological significance of neurotrophin binding to p75NTR in cells that also express Trk receptors has been difficult to ascertain. In the absence of TrkA, NGF binding to p75NGR activates the transcription factor nuclear factor kappa B (NF-kappa B) in rat Schwann cells. This activation is not observed in Schwann cells isolated from mice that lacked p75NTR. The effect was selective for NGF; NF-kappa B was not activated by BDNF or NT-3 (Carter, 1996).

Nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 (NT-3), and NT-5 stimulate sphinomyelin hydrolysis with similar kinetics in p75NTR-NIH-3T3 cells. Although brain-derived neurotrophic factor is slightly more potent than NGF at inducing sphingomyelin hydrolysis, NT-3 and NT-5 induce significant hydrolysis in p75NTR transduced NIH-3T3 cells. NT-3 does not induce sphingomyelin hydrolysis in Trk C transduced NIH-3T3 cells nor in cells expressing a mutated p75NTR containing a 57-amino acid cytoplasmic deletion, thus demonstrating the role of p75NTR in this signal transduction pathway. In p75NTR transduced NIH-3T3 cells, neurotrophin-induces sphingomyelin hydrolysis 1) localizes to an internal pool of sphingomyelin, 2) is not a consequence of receptor internalization, and 3) shows no specificity with respect to the molecular species of sphingomyelin hydrolyzed. In contrast to cells expressing solely p75NTR, NGF (100 ng/ml) does not induce sphingomyelin hydrolysis in PC12 cells. Interestingly, NT-3 induces the same extent of sphingomyelin hydrolysis in PC12 cells as is apparent in p75NTR transcduced NIH-3T3 cells. However, in the presence of NGF, NT-3 is unable to induce sphingomyelin hydrolysis, raising the possibility that Trk modulates p75NTR-dependent sphingomyelin hydrolysis. Inhibition of Trk tyrosine kinase activity with 200 nM K252a enables both NGF and NT-3 in the presence of NGF to induce sphingomyelin hydrolysis. These data support the idea that p75NTR serves as a common signaling receptor for neurotrophins through induction of sphingomyelin hydrolysis and that crosstalk pathways exist between Trk and p75NTR-dependent signaling pathways (Dobrowsky, 1995).

TrkB belongs to the Trk family of tyrosine kinase receptors and mediates the response to brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Both truncated and full-length forms of TrkB receptors are expressed in developing cerebellar granule neurons. BDNF and NT-4/5 increased the survival of cultured cerebellar granule neurons. BDNF and NT-4/5 also induced an autophosphorylation of TrkB receptors and subsequently resulted in a phosphorylation and binding of phospholipase C-gamma (PLC-gamma) and SH2-containing sequence to the autophosphorylated TrkB receptors. Both contain src homology 2 (SH2) regions. In keeping with a signaling function of PLC-gamma, BDNF increased the phosphatidylinositol (PI) turnover and elevated intracellular calcium levels. Cerebellar PKC (See Drosophila PKC) is activated after BDNF or TPA treatment. Survival-promoting effects of BDNF and TPA are blocked with calphostin C, a specific PKC inhibitor. In addition, BDNF activates c-ras in a concentration-dependent manner. These results suggest that two different pathways, the c-ras and the PLC-gamma pathway, are activated by TrkB receptors in primary neurons and that PKC activation is involved in the survival promoting effect of BDNF (Zirrgiebel, 1995).

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, regulates survival and apoptosis of several neuronal populations. These effects are initiated by high-affinity membrane receptors displaying tyrosine kinase activity (trk). BDNF stimulates AP1 binding activity in primary cerebellar neurons. This binding corresponds to a functional complex as it is associated with the induction of AP1-dependent transactivation. Application of AP1 partner mRNAs shows an increase in levels of c-fos and c-jun mRNAs after BDNF treatment, resulting from an induction of their promoters. The cis-acting elements by which BDNF stimulates c-fos transcription were further studied. BDNF impinges on multiple regulatory elements, including the serum-responsive element, Fos AP1-like element, and cyclic AMP (cAMP)-responsive element (CRE) sequences. The latter was stimulated without any detectable increase in cAMP or Ca2+ levels. To confirm that BDNF induces c-fos transcription independently of the protein kinase A/cAMP pathway, a dominant inhibitory mutant of the regulatory subunit of protein kinase A was transfected. The overexpression of this mutant does not affect the c-fos promoter transactivation by BDNF. Thus BDNF stimulates AP1- and CRE-dependent transcription through a mechanism that is distinct from the cAMP- and Ca(2+)-dependent pathways in CNS neurons (Gaiddon, 1996).

The TrkA receptor protein tyrosine kinase is involved in signalling PC12 cell differentiation and cessation of cell division in response to nerve growth factor (NGF). To assess the importance of adaptor proteins and Ras in NGF control of phosphoinositide 3-OH kinase (PI 3- kinase), specific receptor mutations in Trk have been employed. Phosphorylation of tyrosine 490, but not 785, of Trk is essential for activation of both Ras and PI 3-kinase in vivo, correlating with tyrosine phosphorylation of Shc and binding of Shc to the adaptor Grb2 and the Ras exchange factor Sos. A mutant receptor that lacks Y490 and Y785, but contains an introduced YxxM motif that binds the regulatory domain of PI 3-kinase, is unable to activate Ras despite causing increased PI 3-kinase activity. This indicates clearly that activation of PI 3-kinase by itself is not sufficient to cause activation of Ras, arguing against a model in which PI 3-kinase acts upstream of Ras. The Shc site of Trk is thus crucial for the activation of Ras and PI 3-kinase (Hallberg, 1998).

Nerve growth factor (NGF) and other neurotrophins support survival of neurons through processes that are incompletely understood. The transcription factor CREB is a critical mediator of NGF-dependent gene expression, but whether CREB family transcription factors regulate expression of genes that contribute to NGF-dependent survival of sympathetic neurons is unknown. To determine whether CREB-mediated gene expression is necessary for NGF-dependent neuronal survival, this study monitored survival of sympathetic neurons after expression of either of two distinct inhibitors of CREB. One CREB inhibitor, A-CREB, is a potent and selective inhibitor of CREB DNA binding activity. The other, CREBm1, binds to CREB binding sites in DNA but is not activated because the transcriptional regulatory residue, serine 133, is mutated to alanine. CREB-mediated gene expression is both necessary for NGF-dependent survival and sufficient on its own to promote survival of sympathetic neurons. Moreover, expression of Bcl-2 is activated by NGF and other neurotrophins by a CREB-dependent transcriptional mechanism. A region of the bcl-2 gene between 1640 and 1337 relative to the translation start site is required for NGF-sensitive transcription. This region contains a near-perfect consensus CRE. Activated CREB can bind to this region of the bcl-2 promoter, and this interaction is critical for expression of Bcl-2 in a B lymphocyte cell line. Thus, a test was performed to see if the integrity of the bcl-2 CRE is necessary for the NGF-induced expression of bcl-2. A bcl-2 reporter construct harboring a two-base pair mutation of the CRE, rendering it unable to bind CREB, is impaired in its responsiveness to NGF. Overexpression of Bcl-2 reduces the death-promoting effects of CREB inhibition. Together, these data support a model in which neurotrophins promote survival of neurons, in part through a mechanism involving CREB family transcription factor-dependent expression of genes encoding prosurvival factors (Riccio, 1999).

The TrkB protein tyrosine kinase is a high affinity receptor for brain derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). TrkB autophosphorylation occurs on five cytoplasmic tyrosines: Y484, Y670, Y674, Y675, and Y785. Using site directed mutagenesis, the importance of TrkB tyrosines 484 and 785 have been assessed in affecting TrkB-mediated signaling events leading to NIH 3T3 cell mitogenesis and survival. Mutation of TrkB tyrosine 484, while having no affect on BDNF-inducible PLCgamma and Cbl tyrosine phosphorylation, is essential for the phosphorylation of Shc, the complete activation of extracellular regulated kinase 1/2 (ERK1/2) and the induction of c-fos protein synthesis. In contrast, mutation of Y785 does not significantly affect BDNF-inducible Shc phosphorylation, ERK1/2 activation, or c-fos protein synthesis, but completely inhibits the tyrosine phosphorylation of PLCgamma and Cbl. These data indicate that both ERK-dependent and ERK-independent signaling pathways lead to BDNF-inducible mitogenesis and survival (McCarty, 1999).

A human cDNA for the signaling adapter molecule FRS-2/suc1-associated neurotrophic factor target has been isolated and has been shown to be tyrosine-phosphorylated in response to nerve growth factor (NGF) stimulation. Importantly, the phosphotyrosine binding domain of FRS-2 directly binds the Trk receptors at the same phosphotyrosine residue that binds the signaling adapter Shc, suggesting a model in which competitive binding between FRS-2 and Shc regulates differentiation versus proliferation. Consistent with this model, FRS-2 binds Grb-2, Crk, the SH2 domain containing tyrosine phosphatase SH-PTP-2, the cyclin-dependent kinase substrate p13(suc1), and the Src homology 3 (SH3) domain of Src, providing a functional link between TrkA, cell cycle, and multiple NGF signaling effectors. Importantly, overexpression of FRS-2 in cells expressing an NGF nonresponsive TrkA receptor mutant reconstitutes the ability of NGF to stop cell cycle progression and to stimulate neuronal differentiation (Meakin, 1999).

Neurotrophins influence growth and survival of specific populations of neurons through activation of Trks, members of the receptor tyrosine kinase (RTK) family. The identification and characterization of two substrates of Trk kinases, rAPS and SH2-B, are described, which are closely related Src homolog 2 (SH2) domain-containing signaling molecules. rAPS and SH2-B are substrates of TrkB and TrkC in cortical neurons and SH2-B is a substrate of TrkA in sympathetic neurons. Moreover, rAPS and SH2-B bind to Grb2, and both are sufficient to mediate NGF induction of Ras, MAP kinase (MAPK), and morphological differentiation of PC12 cells. Lastly, antibody perturbation and transient transfection experiments indicate that SH2-B, or a closely related molecule, is necessary for NGF-dependent signaling in neonatal sympathetic neurons. Together, these observations indicate that rAPS and SH2-B mediate Trk signaling in developing neurons (Qian, 1998).

To determine how signals emanating from Trk transmit neurotrophin actions in primary neurons, an examination was made of the ability of TrkB mutated at defined effector binding sites to promote sympathetic neuron survival or local axon growth. TrkB stimulates signaling proteins and induces survival and growth in a manner similar to TrkA. TrkB mutated at the Shc binding site supports survival and growth poorly, relative to wild-type TrkB, whereas TrkB mutated at the PLC-gamma1 binding site supports growth and survival well. TrkB-mediated neuronal survival is dependent on PI3-kinase and to a lesser extent MEK activity, while growth depends upon both MEK and PI3-kinase activities. These results indicate that the TrkB-Shc site mediates both neuronal survival and axonal outgrowth by activating the PI3-kinase and MEK signaling pathways (Atwall, 2000).

Thus, the Shc binding site in TrkB regulates the majority of sympathetic neuron survival and local axon growth. In the absence of this site, survival is reduced to 18% and growth reduced to ~30%, as compared to wild type TrkB-expressing neurons. No other single mutation in Trk significantly affects survival or growth in this manner. Mutation of the PLC-gamma1 binding site in combination with the Shc site completely suppresses survival but does not further reduce growth responses. It has been shown that in TrkBShc-/Shc- mice, the Shc site controls a small portion of BDNF-dependent survival (25% loss in the vestibular ganglion and no loss of BDNF-dependent nodose neurons), and the majority of NT-4-dependent survival in vivo. The Shc site is also essential for the survival in culture of nodose neurons in response to either BDNF or NT-4. Together with the data presented here, these results suggest that TrkB mediates survival largely via signaling through the Shc binding site. The discrepancy between in vivo and in vitro data for BDNF-dependent neuronal populations may arise because, in vivo, other growth factors may collaborate with BDNF-bound TrkB Shc- to compensate for the decrease in TrkB signaling through this site. Since BDNF has been shown to be a better ligand than NT-4 for TrkB Shc-, perhaps BDNF-dependent populations are better able to benefit from such compensation in vivo (Atwall, 2000 and references therein).

There have been few studies examining the role of Trk signaling pathways in the promotion and regulation of axonal growth. This issue has been examined using dissociated Xenopus spinal cultures from day-old embryos following blastomere injection with mRNA for wild-type or mutant Trk isoforms. One conclusion from that study was that PLC-gamma activation is essential for axon elongation. The results presented here using mammalian sympathetic neurons are very different. The TrkB PLC-gamma1- mutant, which is defective in activating PLC-gamma1 as assessed by tyrosine phosphorylation, displays no deficits in axon elongation in sympathetic neurons. Furthermore, growth promoted by TrkB encoding both Shc and PLC-gamma1 site mutations is no worse than by TrkB encoding only the Shc site mutation, further confirming that the PLC-gamma1 site plays no role in axon growth in these cultures. It has also been concluded that the PLC-gamma and PI3-kinase sites are both essential for local growth cone turning responses in Xenopus spinal neurons. Conversely, the Shc site is the most important for mediating local axon growth in sympathetic neurons. Thus, the data presented here, combining genetic and biochemical analysis, provides conclusive evidence that signaling through the Shc site mediates the majority of neurotrophin-dependent local axon growth (Atwall, 2000 and references therein).

How does the Shc site mediate survival and axon outgrowth? Whereas mutation of the Shc site clearly eliminates Shc tyrosine phosphorylation, this mutation can effect other signaling processes such as FRS-2 binding to Trk. Thus, it cannot be conclusively said that Shc itself is required for these biological effects. However, the MEK/ERK and PI3-kinase/Akt (see Drosophila Akt1 pathways, both of which are likely dependent on Shc signaling through Ras, are essential for survival and local growth. TrkB mutated at the Shc site does retain a limited ability to stimulate axonal growth and induce ERK and Akt phosphorylation in response to BDNF. This may reflect the ability of Trk to use adaptor proteins other than Shc to stimulate these events. For example, TrkA mutated at the Shc site can activate ERK through the PLC-gamma1 site. TrkA also can stimulate ERK activity independently of the Shc site via rAPS and SH2-B, Akt via Gab1, and neuritogenesis in PC12 cells via SNT. Signaling proteins such as these whose activities are regulated independently of the Shc site may account for the residual 30% of axonal growth observed in neurons expressing TrkB Shc site mutants (Atwall, 2000 and references therein).

No report has described the signals involved in promoting local axon outgrowth. NGF promotes locally increased axonal growth in culture and increased target innervation in vivo. Attempts to address the intracellular signaling mechanisms that regulate such local responses have used systems where global or local affects on growth cannot be distinguished, and where survival and growth effects cannot be segregated. Here, using compartmented cultures, which overcome both of these confounding local issues, it was found that local TrkB-mediated axon elongation is dependent on both PI3-kinase and MEK activity, as activated via the Shc site. How do MEK and PI3-kinase promote local axonal growth? The MEK/ERK pathway is well known to phosphorylate microtubule-associated proteins (MAPs), including Tau, that regulate microtubule stability and control axonal elongation. ERKs also phosphorylate neurofilament proteins. Similarly, PI3-kinase regulates or associates with a number of cytoskeletal proteins, including actin, tubulin, and actin-regulating proteins. The studies presented here suggest that both the MEK/ERK and PI3-kinase pathways are ideally positioned to regulate local growth events ranging from the directionality of growth cone extension to axon elongation itself (Atwall, 2000 and references therein).

Studies reported here also demonstrate that the activities of PI3-kinase, and to a lesser extent MEK, are involved in regulating TrkB-induced sympathetic neuron survival. Evidence for the role of the PI3-kinase/Akt pathway in NGF-mediated survival of peripheral neurons is well documented. PI3-kinase/Akt regulates survival by inhibiting the activities of the cell death proteins Bad and the transcription factor Forkhead in cerebellar neurons and by suppressing the JNK/p53 cell death pathway in sympathetic neurons. In contrast to PI3-kinase/Akt, MEK activity is not important for regulating NGF-dependent peripheral neuron survival. However, MEK activity can mediate, to some extent, cell survival in neurotrophin-regulated neuronal systems, including the survival of cultured cerebellar granule neurons and retinal ganglion cells, and neuroprotection against camptothecin-induced death of cortical neurons and CA-induced death of sympathetic neurons. In addition, sympathetic neuronal survival promoted by activated Ras occurs partly through the MEK/ERK pathway. MEK, together with Akt, may regulate survival by activating the transcription factor CREB, which is a critical regulator of NGF-mediated neuronal survival of sympathetic neurons and of BDNF-mediated survival of cerebellar neurons (Atwall, 2000 and references therein).

In general, TrkA uses PI3-kinase activity to regulate cell survival, while TrkB uses both PI3-kinase and MEK. TrkB thus appears to function in a more 'flexible' manner, using multiple signaling pathways to control specific effects. TrkB may require such signaling flexibility to converge and synergize with many divergent survival cues that CNS neurons are exposed to. Whether such distinctions between TrkA and TrkB generalize to other neuronal responses such as growth, phenotypic modulation, or ongoing plasticity remains to be determined. Nevertheless, it may well be that TrkA may only have to make use of a single signaling pathway such as PI3-kinase to induce responses such as survival, suggesting that TrkA and TrkB are fundamentally different in their signaling capabilities (Atwall, 2000 and references therein).

Trk receptors and synaptic function

In Xenopus nerve-muscle cocultures, BDNF and NT-3, but not NGF, elicite significant changes in several properties of spontaneous synaptic currents (SSCs), indicative of more mature synapses. Most synapses treated by the neurotrophins exhibited a bell-shaped distribution of SSC amplitudes, which reflects mature quantal secretion. The neurotrophins also potentiate the efficacy and reliability of stimulus-induced synaptic transmission. Moreover, BDNF and NT-3 increase the levels of the synaptic vesicle proteins, synaptophysin, and synapsin 1 in the spinal neurons. The number of varicosities per neuron also show a significant increase after neurotrophin treatment. The effects of the neurotrophins appear to be mediated by the Trk family of receptor tyrosine kinases, primarily through a presynaptic mechanism. These results suggest that BDNF and NT-3 promote functional maturation of synapses (Wang, 1995).

Flux of signalling endosomes undergoing axonal retrograde transport is encoded by presynaptic activity and TrkB

Axonal retrograde transport of signalling endosomes from the nerve terminal to the soma underpins survival. As each signalling endosome carries a quantal amount of activated receptors, it was hypothesized that it is the frequency of endosomes reaching the soma that determines the scale of the trophic signal. This study shows that upregulating synaptic activity markedly increased the flux of plasma membrane-derived retrograde endosomes (labelled using cholera toxin subunit-B: CTB) in hippocampal neurons cultured in microfluidic devices, and live Drosophila larval motor neurons. Electron and super-resolution microscopy analyses revealed that the fast-moving sub-diffraction-limited CTB carriers contained the TrkB neurotrophin receptor (see Drosophila Ror), transiently activated by synaptic activity in a BDNF-independent manner. Pharmacological and genetic inhibition of TrkB activation selectively prevented the coupling between synaptic activity and the retrograde flux of signalling endosomes. TrkB activity therefore controls the encoding of synaptic activity experienced by nerve terminals, digitalized as the flux of retrogradely transported signalling endosomes (Wang, 2016).

Trk receptors and retrograde transport of ligand

The receptors involved in retrograde transport of neurotrophins from the retina to the isthmo-optic nucleus (ION) of chick embryos were characterized using antibodies to the p75 neurotrophin receptor and trkB receptors. Survival of neurons in the ION has been shown previously to be regulated by target-derived trophic factors with survival promoted or inhibited by ocular injection of brain-derived neurotrophic factor (BDNF) or nerve growth factor (NGF), respectively. During the period of target dependence, these neurons express trkB and p75 neurotrophin receptor but not trkA or trkC mRNAs. BDNF and NT-3 are transported efficiently at low doses, whereas NGF is transported significantly only at higher doses. The transport of BDNF and NT-3 is reduced by high concentrations of NGF or by antibodies to either trkB or the p75 neurotrophin receptor. Thus both receptors help mediate retrograde transport of these neurotrophins. Ocular injection of the comparatively specific trk inhibitor K252a does not reduce transport of exogenous BDNF, but does induce significant neuronal death in the ION, which could not be prevented by co-injection of BDNF. Thus, transport of BDNF alone does not generate a trophic signal at the cell body when axonal trkB is inactivated. In summary, these results indicate that both p75 neurotrophin and trkB receptors can mediate internalization and retrograde transport of BDNF, but activation of trkB seems to be essential for the survival-promoting actions of this neurotrophin (von Bartheld, 1996).

Nerve growth factor (NGF) is a neurotrophic factor secreted by cells that are the targets of innervation of sympathetic and some sensory neurons. However, what remains unclear is the mechanism by which the NGF signal is propagated from the axon terminal to the cell body (which can be more than 1 meter away). This mechanism influences biochemical events critical for growth and survival of neurons. An NGF-mediated signal transmitted from the terminals and distal axons of cultured rat sympathetic neurons to their nuclei regulates phosphorylation of the transcription factor CREB (cyclic adenosine monophosphate response element-binding protein). Internalization of NGF and its receptor tyrosine kinase TrkA, and the transport of both to the cell body, are required for transmission of this signal. The tyrosine kinase activity of TrkA is required to maintain TrkA in an autophosphorylated state upon its arrival in the cell body and for propagation of the signal to CREB within neuronal nuclei. Thus, an NGF-TrkA complex is a messenger that delivers the NGF signal from axon terminals to cell bodies of sympathetic neurons (Riccio, 1997).

Developmental expression and function of Trk receptors

Antibodies to trkC, the major receptor for NT-3, were used to examine trkC expression and function during the formation and maturation of the chick dorsal root ganglion (DRG). In the immature DRG, the majority of cells express trkC, and inhibition of trkC activation results in reductions in neuronal numbers before the period of target-mediated cell death, the time when neurotrophins previously have been shown to regulate survival. Furthermore, blockade of trkC in ovo induced reductions in subpopulations of DRG neurons known to be dependent on NGF, in addition to those dependent on NT-3 during the target-regulated cell death period. There is an early function for NT-3 on immature DRG neurons. Whereas BDNF and NGF can support a subset of immature DRG neurons in vitro, activation of the trkC receptor either by NT-3 binding or via antibody-mediated cross-linking induces the most robust survival response. When all three neurotrophins are combined, the number of surviving neurons does not exceed that supported by NT-3 alone. Together, these data are consistent with coexpression of more than one trk receptor family member on immature sensory neurons, and they demonstrate that inhibition of trkC activation has surprisingly early and pleiotrophic effects on the development of spinal sensory ganglia (Lefcort, 1995).

Double mutant mice deficient in pairs of two different Trk receptors were generated and analysed the effects on survival and differentiation of dorsal root ganglion (DRG), inner ear cochlear and vestibular sensory neurons. In most combinations of mutant trk alleles, the defects observed in double compared to single mutant mice are additive. However, double homozygous trkA-/-;trkB-/- DRG and trkB-/-;trkC-/- vestibular neurons show the same degree of survival as single trkA-/- and trkB-/- mice, respectively, suggesting that those neurons require both Trk signaling pathways for survival. In situ hybridisation analysis of DRG neurons of double mutant mice revealed differential expression of excitatory neuropeptides. Whereas calcitonin-gene-related peptide expression correlates with the trkA phenotype, substance P expression is detected in all combinations of double mutant mice. In the inner ear, TrkB- and TrkC-dependent neurons are at least partially depend on each other for survival, most likely indirectly due to abnormal development of their common targets. This effect is not observed in DRGs, where neurons depending on different Trk receptors generally innervate different targets (Minichiello, 1995).

Neurotrophins are a family of soluble ligands that promote the survival and differentiation of peripheral and central neurons and regulate synaptic function. The two neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4), bind and activate a single high-affinity receptor, TrkB. Experiments in cell culture have revealed that an intact Shc adaptor binding site on TrkB and subsequent activation of the Ras/MAPK pathway are important for neuronal survival and neurite outgrowth. To elucidate the intracellular signaling pathways that mediate the diverse effects of BDNF and NT4 in vivo, the Shc binding site in the trkB gene has been mutated. This trkBshc mutation reveals distinctive responses to BDNF and NT4. While nearly all NT4-dependent sensory neurons are lost in trkBshc mutant mice, BDNF-dependent neurons are modestly affected. Activation of MAP kinases and in vitro survival of cultured trkBshc neurons are reduced in response to both neurotrophins, with NT4 being less potent than BDNF, suggesting differential activation of TrkB by the two ligands. Moreover, while the Ras/MAPK pathway is required for in vitro differentiation of neuronal cells, trkBshc mutant mice do not show any defects in BDNF-dependent differentiation of CNS neurons or in the function of sensory neurons that mediate innocuous touch (Minichiello, 1998).

The neurotrophin survival dependence of peripheral neurons in vitro is regulated by the proapoptotic BCL-2 homolog BAX. To study peripheral neuron development in the absence of neurotrophin signaling, mice were generated that are double null for BAX and nerve growth factor (NGF), and BAX and the NGF receptor TrkA. All dorsal root ganglion (DRG) neurons that normally die in the absence of NGF/TrkA signaling survive if BAX is also eliminated. These neurons extend axons through the dorsal roots and collateral branches into the dorsal horn. In contrast, superficial cutaneous innervation is absent. Furthermore, rescued sensory neurons fail to express biochemical markers characteristic of the nociceptive phenotype. These findings establish that NGF/TrkA signaling regulates peripheral target field innervation and is required for the full phenotypic differentiation of sensory neurons (Patel, 2000).

NGF is a target-derived growth factor for developing sympathetic neurons. Application of NGF exclusively to distal axons of sympathetic neurons leads to an increase in PI3-K signaling in both distal axons and cell bodies. In addition, there is a more critical dependence on PI3-K for survival of neurons supported by NGF acting exclusively on distal axons as compared to neurons supported by NGF acting directly on cell bodies. Interestingly, PI3-K signaling within both cell bodies and distal axons contributes to survival of neurons. The requirement of PI3-K signaling in distal axons for survival may be explained by the finding that inhibition of PI3-K in the distal axons attenuates retrograde signaling. Therefore, a single TrkA effector, PI3-K, has multiple roles within spatially distinct cellular locales during retrograde NGF signaling (Kuruvilla, 2000).

Dissociated sympathetic neurons obtained from newborn rat superior cervical ganglia and grown in compartmentalized cultures were to assess the subcellular distribution and state of activation of PI3-K and its downstream effector Akt (protein kinase B). Neurons were maintained under conditions in which cell bodies and proximal axons (hereafter referred to as the cell body compartment) were exposed to medium containing a neutralizing antibody directed against NGF (alpha-NGF), while distal axons, which are >1 mm away from cell bodies, were exposed to medium containing NGF. These conditions resemble in vivo conditions in which neurons are maintained by NGF acting exclusively on distal axons (Kuruvilla, 2000).

It was asked whether binding of NGF to receptors exclusively on distal axons regulates the activities of PI3-K and Akt in distal axons and/or cell bodies. For these experiments, NGF was removed from medium bathing distal axons for 24 hr. Then, distal axons were exposed to the same medium (control) or medium containing NGF for various times. The activation states of TrkA and Akt were assessed in extracts prepared from cell body and distal axon compartments by immunoblotting using antibodies that recognize the activated, phosphorylated forms of these proteins. P-Trk (Y490) antibodies recognize TrkA when phosphorylated on Tyr-490, which is the Shc recognition site. P-Akt antibodies recognize Akt when phosphorylated on Ser-473, which is necessary for its catalytic activity. Application of NGF to distal axons results in increased levels of P-TrkA (Y490) and P-Akt within distal axons, which are maximal after 20 min. Increases in both P-TrkA (Y490) and P-Akt are also detected in cell bodies but with slower kinetics. A small but reproducible increase in both P-TrkA (Y490) and P-Akt is detected in extracts of cell bodies within 20 min, and a more robust increase is seen at 8 hr. The appearance of P-TrkA (Y490) and P-Akt in both distal axons and cell bodies is coincident with the appearance of PI3-K activity associated with phosphotyrosine immunoprecipitates. Additionally, withdrawal of NGF from distal axons of neurons, which had been grown with medium containing a high concentration of NGF (100 ng/ml) on distal axons and alpha-NGF on cell bodies, leads to a decrease in the levels of both P-TrkA (Y490) and P-Akt in distal axons and in cell bodies. Thus, NGF acting on TrkA receptors on distal axons regulates the phosphorylation/activation of TrkA, PI3-K, and Akt both locally within distal axons and retrogradely to proximal axons and cell bodies of sympathetic neurons (Kuruvilla, 2000).

These results support the idea that PI3-K signaling within both cell bodies and distal axons is necessary for survival of neurons supported by NGF acting on distal axons. Moreover, the requirement of PI3-K signaling in distal axons is more apparent when a submaximal concentration of NGF is used to support survival. How does PI3-K signaling within distal axons contribute to survival? It was found that PI3-K activity in distal axons controls retrograde NGF transport and retrograde signaling, which may be critical for survival. Complete inhibition of PI3-K in distal axons, as assessed by levels of P-Akt, attenuates retrograde transport of NGF by ~80% in two compartment chambers and 65% in three compartment chambers. Thus, there is a small but significant amount of retrograde transport that occurs in a PI3-K-independent manner. These observations may account for the finding that inhibition of PI3-K in distal axons has more dire consequences for neurons supported by 0.5 ng/ml NGF acting on distal axons than for those supported by 50 ng/ml NGF acting on distal axons. Neurons grown in a low, submaximal concentration of NGF are more vulnerable than neurons supported by a high concentration of NGF to a 65%-80% reduction in retrograde signaling (Kuruvilla, 2000). The precise role of PI3-K signaling in distal axons for ligand-dependent internalization, retrograde transport, and retrograde signaling is not clear. It is possible that products of the PI3-K catalyzed reaction are critical for the ligand-dependent production of clathrin-coated pits, into which NGF and TrkA are initially internalized. In support of this idea, there is an essential role for the pleckstrin homology (PH) domain of the GTPase dynamin for receptor-mediated endocytosis. Further, dynamin is required for retrograde transport of NGF in sympathetic neurons. Since the dynamin PH domain binds to phosphoinositide products of the PI3-K-catalyzed reaction, PI3-K activity associated with TrkA may be critical for recruitment of dynamin to regions of the plasma membrane destined to invaginate to form NGF/TrkA-containing clathrin-coated signaling organelles. Similarly, AP-2, which is involved in clathrin coat formation and vesicle sorting at the plasma membrane, contains an amino-terminal phosphoinositide binding domain that is required for its targeting to the plasma membrane. Thus, it is tempting to speculate that PI3-K signaling in distal axons is needed for survival because this TrkA effector controls membrane recruitment of key regulators of NGF/TrkA endocytosis and retrograde TrkA signaling (Kuruvilla, 2000).

If PI3-K in distal axons is required for retrograde signaling, what is the role of PI3-K in cell bodies in neurons supported by NGF acting exclusively on distal axons? Inhibition of PI3-K in cell bodies leads to near complete apoptosis of neurons within 48 hr, but inhibition of PI3-K exclusively in cell bodies does not affect retrograde transport of NGF. Under these conditions, P-Akt in cell bodies is completely blocked, but levels of P-Akt in distal axons are unaffected. These observations indicate that PI3-K and Akt signaling in distal axons alone cannot support neuronal survival. Since constitutively active PI3-K and Akt can support survival of sympathetic neurons, it is speculated that PI3-K signaling in cell bodies is necessary for survival because it supports Akt signaling and phosphorylation of Akt substrates that mediate the prosurvival effects of PI3-K. Indeed, it seems likely that many of the substrates of Akt function, at least in part, within cell bodies. Substrates of Akt include BAD, caspase-9, IKK, the transcription factor forkhead, and, possibly, CREB. By extension, these data support the idea that phosphorylation of Akt substrates within distal axons cannot support neuronal survival. This may be because critical substrates of Akt are either not present in distal axons or that they are present in distal axons but cannot move in the phosphorylated forms from distal axons to cell bodies to affect the apoptotic machinery. P-Akt itself does not move from distal axons to cell bodies to an appreciable extent so the same is likely to be true for products of Akt-catalyzed phosphorylation reactions (Kuruvilla, 2000).

Growth factor signal transduction mechanisms in neurons are arguably more complex than in most other cell types due to the striking morphological specializations of neurons. Most neurons have long axons that can extend centimeters or even one meter from their cell bodies, and target-derived growth factor signals must be propagated over long distances to influence survival and gene expression within cell bodies. These retrograde signals must be integrated with signals coming from dendrites and those emanating from the membrane of the cell body itself. The present study shows that the same NGF effector pathway, the PI3-K pathway, can have different functions in distinct parts of the same neuron during long-range retrograde signaling. Interestingly, the activity of the PI3-K signaling in distal axons indirectly regulates TrkA signaling pathways in cell bodies, including the PI3-K effector pathway. Thus, there exists interdependence of TrkA effector pathways in distinct cellular locales whereby ligand-dependent TrkA effector signaling in one compartment, the distal axon, controls effector signaling in another, the cell body (Kuruvilla, 2000).

Ror: Biological Overview | Developmental Biology | References

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