Wnt signalling requires MTM-6 and MTM-9 myotubularin lipid-phosphatase function in Wnt-producing cells

Wnt proteins are lipid-modified glycoproteins that have important roles in development, adult tissue homeostasis and disease. Secretion of Wnt proteins from producing cells is mediated by the Wnt-binding protein MIG-14/Wls, which binds Wnt in the Golgi network and transports it to the cell surface for release. It has recently been shown that recycling of MIG-14/Wls from the plasma membrane to the trans-Golgi network is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is still poorly understood. In this study, we report the identification of MTM-6 and MTM-9 as novel regulators of MIG-14/Wls trafficking in Caenorhabditis elegans. MTM-6 and MTM-9 are myotubularin lipid phosphatases that function as a complex to dephosphorylate phosphatidylinositol-3-phosphate, a central regulator of endosomal trafficking. We show that mutation of mtm-6 or mtm-9 leads to defects in several Wnt-dependent processes and demonstrate that MTM-6 is required in Wnt-producing cells as part of the MIG-14/Wls-recycling pathway. This function is evolutionarily conserved, as the MTM-6 orthologue DMtm6 is required for Wls stability and Wg secretion in Drosophila. We conclude that regulation of endosomal trafficking by the MTM-6/MTM-9 myotubularin complex is required for the retromer-dependent recycling of MIG-14/Wls and Wnt secretion.     

A Wnt signaling system that specifies two patterns of cell migration in C. elegans

In C. elegans, a bilateral pair of neuroblasts, QL and QR, give rise to cells that migrate in opposite directions along the anteroposterior (A/P) body axis. QL and its descendants migrate posteriorly whereas QR and its descendants migrate anteriorly. We find that a Wnt family member, EGL-20, acts in a dose-dependent manner to specify these opposite migratory behaviors. High levels of EGL-20 promote posterior migration by activating a canonical Wnt signal transduction pathway, whereas low levels promote anterior migration by activating a separate, undefined pathway. We find that the two Q cells respond differently to EGL-20 because they have different response thresholds. Thus, in this system two distinct dose-dependent responses are specified not by graded levels of the Wnt signal, but instead by left-right asymmetrical differences in the cellular responsiveness to Wnt signaling.     

Caenorhabditis elegans Myotubularin MTM-1 Negatively Regulates the Engulfment of Apoptotic Cells

During programmed cell death, apoptotic cells are recognized and rapidly engulfed by phagocytes. Although a number of genes have been identified that promote cell corpse engulfment, it is not well understood how phagocytosis of apoptotic cells is negatively regulated. Here we have identified Caenorhabditis elegans myotubularin MTM-1 as a negative regulator of cell corpse engulfment. Myotubularins (MTMs) constitute a large, highly conserved family of lipid phosphatases. MTM gene mutations are associated with various human diseases, but the cellular functions of MTM proteins are not clearly defined. We found that inactivation of MTM-1 caused significant reduction in cell corpses in strong loss-of-function mutants of ced-1, ced-6, ced-7, and ced-2, but not in animals deficient in the ced-5, ced-12, or ced-10 genes. In contrast, overexpression of MTM-1 resulted in accumulation of cell corpses. This effect is dependent on the lipid phosphatase activity of MTM-1. We show that loss of mtm-1 function accelerates the clearance of cell corpses by promoting their internalization. Importantly, the reduction of cell corpses caused by mtm-1 RNAi not only requires the activities of CED-5, CED-12, and CED-10, but also needs the functions of the phosphatidylinositol 3-kinases (PI3Ks) VPS-34 and PIKI-1. We found that MTM-1 localizes to the plasma membrane in several known engulfing cell types and may modulate the level of phosphatidylinositol 3-phosphate (PtdIns(3)P) in vivo. We propose that MTM-1 negatively regulates cell corpse engulfment through the CED-5/CED-12/CED-10 module by dephosphorylating PtdIns(3)P on the plasma membrane.     

Multiple levels of regulation specify the polarity of an asymmetric cell division in C. elegans

Wnt signaling systems play important roles in the generation of cell and tissue polarity during development. We describe a Wnt signaling system that acts in a new way to orient the polarity of an epidermal cell division in C. elegans. In this system, the EGL-20/Wnt signal acts in a permissive fashion to polarize the asymmetric division of a cell called V5. EGL-20 regulates this polarization by counteracting lateral signals from neighboring cells that would otherwise reverse the polarity of the V5 cell division. Our findings indicate that this lateral signaling pathway also involves Wnt pathway components. Overexpression of EGL-20 disrupts both the asymmetry and polarity of lateral epidermal cell divisions all along the anteroposterior (A/P) body axis. Together our findings suggest that multiple, inter-related Wnt signaling systems may act together to polarize asymmetric cell divisions in this tissue.     

Protein kinase CK2 is required for Wntless internalization and Wnt secretion

Wnt proteins are lipid modified signaling molecules that have essential functions in development and adult tissue homeostasis. Secretion of Wnt is mediated by the transmembrane protein Wntless, which binds Wnt and transports it from the endoplasmic reticulum to the cell surface for release. To maintain efficient Wnt secretion, Wntless is recycled back to the Golgi and the endoplasmic reticulum through endocytosis and retromer dependent endosome to Golgi transport. We have previously identified protein kinase CK2 (CK2) in a genome-wide screen for regulators of Wnt signaling in Caenorhabditis elegans. Here, we show that CK2 function is required in Wnt producing cells for Wnt secretion. This function is evolutionarily conserved, as inhibition of CK2 activity interferes with Wnt5a secretion from mammalian cells. Mechanistically, we show that inhibition of CK2 function results in enhanced plasma membrane localization of Wls in C. elegans and mammalian cells, consistent with the notion that CK2 is involved in the regulation of Wls internalization.     

Identification of genes that regulate a left-right asymmetric neuronal migration in Caenorhabditis elegans

In C. elegans, cells of the QL and QR neuroblast lineages migrate with left-right asymmetry; QL and its descendants migrate posteriorly whereas QR and its descendants migrate anteriorly. One key step in generating this asymmetry is the expression of the Hox gene mab-5 in the QL descendants but not in the QR descendants. This asymmetry appears to be coupled to the asymmetric polarizations and movements of QL and QR as they migrate and relies on an asymmetric response to an EGL-20/Wnt signal. To identify genes involved in these complex layers of regulation and to isolate targets of mab-5 that direct posterior migrations, we screened visually for mutants with cell migration defects in the QL and QR lineages. Here, we describe a set of new mutants (qid-5, qid-6, qid-7, and qid-8) that primarily disrupt the migrations of the QL descendants. Most of these mutants were defective in mab-5 expression in the QL lineage and might identify genes that interact directly or indirectly with the EGL-20/Wnt signaling pathway.     

Wnt signals and frizzled activity orient anterior-posterior axon outgrowth in C. elegans

Secreted proteins of the Wnt family affect axon guidance, asymmetric cell division, and cell fate. We show here that C. elegans Wnts acting through Frizzled receptors can shape axon and dendrite trajectories by reversing the anterior-posterior polarity of neurons. In lin-44/Wnt and lin-17/Frizzled mutants, the polarity of the PLM mechanosensory neuron is reversed along the body axis: the long PLM process, PLM growth cone, and synapses are posterior to its cell body instead of anterior. Similarly, the polarity of the ALM mechanosensory neuron is reversed in cwn-1 egl-20 Wnt double mutants, suggesting that different Wnt signals regulate neuronal polarity at different anterior-posterior positions. LIN-17 protein is asymmetrically localized to the posterior process of PLM in a lin-44-dependent manner, indicating that Wnt signaling redistributes LIN-17 in PLM. In this context, Wnts appear to function not as instructive growth cone attractants or repellents, but as organizers of neuronal polarity.     

Wnt-7a modulates the synaptic vesicle cycle and synaptic transmission in hippocampal neurons

Wnt signaling is essential for neuronal development and the maintenance of the developing nervous system. Recent studies indicated that Wnt signaling modulates long term potentiation in adult hippocampal slices. We report here that different Wnt ligands are present in hippocampal neurons of rat embryo and adult rat, including Wnt-4, -5a, -7a, and -11. Wnt-7a acts as a canonical Wnt ligand in rat hippocampal neurons, stimulates clustering of presynaptic proteins, and induces recycling and exocytosis of synaptic vesicles as studied by FM dyes. Wnt-3a has a moderate effect on recycling of synaptic vesicles, and no effect of Wnt-1 and Wnt-5a was detected. Electrophysiological analysis on adult rat hippocampal slices indicates that Wnt-7a, but not Wnt-5a, increases neurotransmitter release in CA3-CA1 synapses by decreasing paired pulse facilitation and increasing the miniature excitatory post-synaptic currents frequency. These results indicate that the presynaptic function of rat hippocampal neurons is modulated by the canonical Wnt signaling.     

The Frizzled family: receptors for multiple signal transduction pathways

Frizzled genes encode integral membrane proteins that function in multiple signal transduction pathways. They have been identified in diverse animals, from sponges to humans. The family is defined by conserved structural features, including seven hydrophobic domains and a cysteine-rich ligand-binding domain. Frizzled proteins are receptors for secreted Wnt proteins, as well as other ligands, and also play a critical role in the regulation of cell polarity. Frizzled genes are essential for embryonic development, tissue and cell polarity, formation of neural synapses, and the regulation of proliferation, and many other processes in developing and adult organisms; mutations in human frizzled-4 have been linked to familial exudative vitreoretinopathy. It is not yet clear how Frizzleds couple to downstream effectors, and this is a focus of intense study.     

Alternative splicing affecting a novel domain in the C. elegans EGL-15 FGF receptor confers functional specificity

Fibroblast growth factor (FGF) receptors trigger a wide variety of cellular responses as diverse as cell migration, cell proliferation and cell differentiation. However, the molecular basis of the specificity of these responses is not well understood. The C. elegans FGF receptor EGL-15 similarly mediates a number of different responses, including transducing a chemoattractive signal and mediating an essential function. Analysis of the migration-specific alleles of egl-15 has identified a novel EGL-15 isoform that provides a molecular explanation for the different phenotypic effects of lesions at this locus. Alternative splicing yields two EGL-15 proteins containing different forms of a domain located within the extracellular region of the receptors immediately after the first IG domain. Neither of these two domain forms is found in any other FGF receptor. We have tested the roles of these EGL-15 receptor isoforms and their two FGF ligands for their signaling specificity. Our analyses demonstrate different physiological functions for the two receptor variants. EGL-15(5A) is required for the response to the FGF chemoattractant that guides the migrating sex myoblasts to their final positions. By contrast, EGL-15(5B) is both necessary and sufficient to elicit the essential function mediated by this receptor.     

Wnt Signaling Is Regulated by Endoplasmic Reticulum Retention

Precise regulation of Wnt signaling is important in many contexts, as in development of the vertebrate forebrain, where excessive or ectopic Wnt signaling leads to severe brain defects. Mutation of the widely expressed oto gene causes loss of the anterior forebrain during mouse embryogenesis. Here we report that oto is the mouse ortholog of the gpi deacylase gene pgap1, and that the endoplasmic reticulum (ER)-resident Oto protein has a novel and deacylase-independent function during Wnt maturation. Oto increases the hydrophobicities of Wnt3a and Wnt1 by promoting the addition of glycophosphatidylinositol (gpi)-like anchors to these Wnts, which results in their retention in the ER. We also report that oto-deficient embryos exhibit prematurely robust Wnt activity in the Wnt1 domain of the early neural plate. We examine the effect of low oto expression on Wnt1 in vitro by knocking down endogenous oto expression in 293 and M14 melanoma cells using shRNA. Knockdown of oto results in increased Wnt1 secretion which is correlated with greatly enhanced canonical Wnt activity. These data indicate that oto deficiency increases Wnt signaling in vivo and in vitro. Finally, we address the mechanism of Oto-mediated Wnt retention under oto-abundant conditions, by cotransfecting Wnt1 with gpi-specific phospholipase D (GPI-PLD). The presence of GPI-PLD in the secretory pathway results in increased secretion of soluble Wnt1, suggesting that the gpi-like anchor lipids on Wnt1 mediate its retention in the ER. These data now provide a mechanistic framework for understanding the forebrain defects in oto mice, and support a role for Oto-mediated Wnt regulation during early brain development. Our work highlights a critical role for ER retention in regulating Wnt signaling in the mouse embryo, and gives insight into the notoriously inefficient secretion of Wnts.     

LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way

Wnt signaling through the canonical beta-catenin pathway plays essential roles in development and disease. Low-density-lipoprotein receptor-related proteins 5 and 6 (Lrp5 and Lrp6) in vertebrates, and their Drosophila ortholog Arrow, are single-span transmembrane proteins that are indispensable for Wnt/beta-catenin signaling, and are likely to act as Wnt co-receptors. This review highlights recent progress and unresolved issues in understanding the function and regulation of Arrow/Lrp5/Lrp6 in Wnt signaling. We discuss Arrow/Lrp5/Lrp6 interactions with Wnt and the Frizzled family of Wnt receptors, and with the intracellular beta-catenin degradation apparatus. We also discuss the regulation of Lrp5/Lrp6 by other extracellular ligands, and LRP5 mutations associated with familial osteoporosis and other disorders.     

Interleukin-1 receptor antagonist inhibits metastatic potential by down-regulating CXCL12/CXCR4 signaling axis in colorectal cancer

Wnt signaling plays key roles in cellular and physiological processes, including cell proliferation, differentiation and migration during development and tissue homeostasis in adults. This pathway can be defined as Wnt/β-catenin-dependent or β-catenin-independent or “non-canonical”, both signaling are involved in neurite and synapse development/maintenance. Porcupine (PORCN), an acylase that o-acylates Wnt ligands, a major modification in secretion and interaction with its receptors. We use Wnt-C59, a specific PORCN inhibitor, to block the secretion of endogenous Wnts in embryonic hippocampal neurons (DIV 4). Under these conditions, the activity of exogenous Wnt ligands on the complexity of the dendritic tree and axonal polarity were evaluated Cultured primary embryonic hippocampal neurons obtained from Sprague–Dawley rat fetuses (E18), were cultured until day in vitro (DIV) 4 (according to Banker´s protocol) and treated with Wnt-C59 for 24 h, Wnt ligands were added to the cultures on DIV 3 for 24 h. Dendritic arbors and neurites were analysis by fluorescence microscopy. Transfection with Lipofectamine 2000 on DIV 2 of plasmid expressing eGFP and KIF5-Cherry was carried out to evaluate neuronal polarity. Immunostaining was performed with MAP1B and Tau protein. Immunoblot analysis was carried out with Wnt3a, β-catenin and GSK-3β (p-Ser9). Quantitative analysis of dendrite morphology was carried out with ImageJ (NIH) software with Neuron J Plugin. We report, here, that Wnt-C59 treatment changed the morphology of the dendritic arbors and neurites of embryonic hippocampal neurons, with decreases β-catenin and Wnt3a and an apparent increase in GSK-3β (p-Ser9) levels. No effect was observed on axonal polarity. In sister cultures, addition of exogenous Wnt3a, 5a and 7a ligands rescued the changes in neuronal morphology. Wnt3a restored the length of neurites to near that of the control, but Wnt7a increased the neurite length beyond that of the control. Wnt5a also restored the length of neurites relative to Wnt concentrations. Results indicated that Wnt ligands, added exogenously, restored dendritic arbor complexity in embryonic hippocampal neurons, previously treated with a high affinity specific Porcupine inhibitor. We proposed that PORCN is an emerging molecular target of interest in the search for preclinical options to study and treat Wnt-related diseases.     

The C. elegans cGMP-Dependent Protein Kinase EGL-4 Regulates Nociceptive Behavioral Sensitivity

Signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. Herein we report a new role for the cGMP-dependent protein kinase EGL-4 in the negative regulation of G protein-coupled nociceptive chemosensory signaling. C. elegans lacking EGL-4 function are hypersensitive in their behavioral response to low concentrations of the bitter tastant quinine and exhibit an elevated calcium flux in the ASH sensory neurons in response to quinine. We provide the first direct evidence for cGMP/PKG function in ASH and propose that ODR-1, GCY-27, GCY-33 and GCY-34 act in a non-cell-autonomous manner to provide cGMP for EGL-4 function in ASH. Our data suggest that activated EGL-4 dampens quinine sensitivity via phosphorylation and activation of the regulator of G protein signaling (RGS) proteins RGS-2 and RGS-3, which in turn downregulate Gα signaling and behavioral sensitivity.     

Wnt Signaling in the Vertebrate Central Nervous System: From Axon Guidance to Synaptic Function

Regulation of cell signaling by Wnt proteins is critical for the formation of neuronal circuits. Wnts modulate axon pathfinding, dendritic development, and synaptic assembly. Through different receptors, Wnts activate diverse signaling pathways that lead to local changes on the cytoskeleton or global cellular changes involving nuclear function. Recently, a link between neuronal activity, essential for the formation and refinement of neuronal connections, and Wnt signaling has been uncovered. Indeed, neuronal activity regulates the release of Wnt and the localization of their receptors. Wnts mediate synaptic structural changes induced by neuronal activity or experience. New emerging evidence suggests that dysfunction in Wnt signaling contributes to neurological disorders. In this article, the attention is focused on the function of Wnt signaling in the formation of neuronal circuits in the vertebrate central nervous system.The formation of neuronal connections requires the navigation of axons to their appropriate synaptic targets, the formation of terminal branches, and the assembly of functional synapses. These processes greatly depend on the proper dialogue between axons and their environment as they navigate to their target, and between axons and their postsynaptic dendrites during synapse assembly. A combination of secreted molecules and transmembrane proteins modulates these processes. Studies over the last 10 years have revealed an essential role for Wnt signaling in axon pathfinding, dendritic development, and synapse assembly in both central and peripheral nervous systems. Wnts also modulate basal synaptic transmission and the structural and functional plasticity of synapses in the central nervous system. Studies of Wnts in the nervous system have significantly contributed to our current understanding of the molecular mechanisms that control neuronal circuit assembly. These studies have also shed light into fundamental aspects of cell signaling such as novel mechanisms of protein secretion (Korkut et al. 2009) and receptor dynamics (Sahores et al. 2010). Here I review the mechanisms by which Wnts modulate axon guidance and synapse formation in the vertebrate central nervous system. I also discuss the increasing evidence in support for a role of Wnts in basal synaptic transmission, synaptic plasticity, and neurological disorders.     

SAX-3 (Robo) and UNC-40 (DCC) Regulate a Directional Bias for Axon Guidance in Response to Multiple Extracellular Cues

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