Dual roles for Wnt signalling during the formation of the vertebrate neuromuscular junction.

Wnt proteins play prominent roles in different aspects of neuronal development culminating with the formation of complex neuronal circuits. Here, we discuss new studies addressing the function of Wnt signalling at the peripheral neuromuscular junction (NMJ). In both, invertebrate and vertebrate organisms, Wnt signalling promotes and also inhibits the assembly of the neuromuscular synapse. Here, we focus our attention on recent studies at the vertebrate NMJ that demonstrate that some Wnt proteins collaborate with the Agrin-MuSK signalling to induce post-synaptic differentiation. In contrast, Wnts that activate the Wnt/ß-catenin signalling inhibit post-synaptic differentiation. The dual function of different Wnts might finely modulate the proper apposition of the pre- and post-synaptic terminals during NMJ formation and growth.

Retrograde signalling at the synapse: a role for Wnt proteins.

The formation of functional synapses requires a proper dialogue between incoming axons and their future synaptic targets. As axons approach their target, they are instructed to slow down and remodel to form proper presynaptic terminals. Although significant progress has been made in the identification of the mechanisms that control axon guidance, little is known about the mechanisms that regulate the conversion of actively growing axon into a presynaptic terminal. We found that Wnt secreted proteins are retrograde signals that regulate the terminal arborization of axons and synaptic differentiation. Wnts released from postsynaptic neurons induce extensive remodelling on incoming axons. This remodelling is manifested by a decrease in axon extension with a concomitant increase in growth-cone size. This morphological change is correlated with changes in the dynamics and organization of microtubules. Studies of a vertebrate synapse and the Drosophila neuromuscular junction suggest that a conserved Wnt signalling pathway modulates presynaptic microtubules as axons remodel during synapse formation. In this paper I discuss the role of the Wnt-Dvl (Dishevelled protein)-GSK-3beta (glycogen synthase kinase-3beta) signalling pathway in axon remodelling during synapse formation in the central nervous system.

Dishevelled-1 regulates microtubule stability: a new function mediated by glycogen synthase kinase-3beta.

Dishevelled has been implicated in the regulation of cell fate decisions, cell polarity, and neuronal function. However, the mechanism of Dishevelled action remains poorly understood. Here we examine the cellular localization and function of the mouse Dishevelled protein, DVL-1. Endogenous DVL-1 colocalizes with axonal microtubules and sediments with brain microtubules. Expression of DVL-1 protects stable microtubules from depolymerization by nocodazole in both dividing cells and differentiated neuroblastoma cells. Deletion analyses reveal that the PDZ domain, but not the DEP domain, of DVL-1 is required for microtubule stabilization. The microtubule stabilizing function of DVL-1 is mimicked by lithium-mediated inhibition of glycogen synthase kinase-3beta (GSK-3beta) and blocked by expression of GSK-3beta. These findings suggest that DVL-1, through GSK-3beta, can regulate microtubule dynamics. This new function of DVL-1 in controlling microtubule stability may have important implications for Dishevelled proteins in regulating cell polarity.

Axonal remodeling and synaptic differentiation in the cerebellum is regulated by WNT-7a signaling.

Synapse formation requires changes in cell morphology and the upregulation and localization of synaptic proteins. In the cerebellum, mossy fibers undergo extensive remodeling as they contact several granule cells and form complex, multisynaptic glomerular rosettes. Here we show that granule cells secrete factors that induce axon and growth cone remodeling in mossy fibers. This effect is blocked by the WNT antagonist, sFRP-1, and mimicked by WNT-7a, which is expressed by granule cells. WNT-7a also induces synapsin I clustering at remodeled areas of mossy fibers, a preliminary step in synaptogenesis. Wnt-7a mutant mice show a delay in the morphological maturation of glomerular rosettes and in the accumulation of synapsin I. We propose that WNT-7a can function as a synaptogenic factor.

Inhibition of cyclin-dependent kinases, GSK-3beta and CK1 by hymenialdisine, a marine sponge constituent.

BACKGROUND: Over 2000 protein kinases regulate cellular functions. Screening for inhibitors of some of these kinases has already yielded some potent and selective compounds with promising potential for the treatment of human diseases. RESULTS: The marine sponge constituent hymenialdisine is a potent inhibitor of cyclin-dependent kinases, glycogen synthase kinase-3beta and casein kinase 1. Hymenialdisine competes with ATP for binding to these kinases. A CDK2-hymenialdisine complex crystal structure shows that three hydrogen bonds link hymenialdisine to the Glu81 and Leu83 residues of CDK2, as observed with other inhibitors. Hymenialdisine inhibits CDK5/p35 in vivo as demonstrated by the lack of phosphorylation/down-regulation of Pak1 kinase in E18 rat cortical neurons, and also inhibits GSK-3 in vivo as shown by the inhibition of MAP-1B phosphorylation. Hymenialdisine also blocks the in vivo phosphorylation of the microtubule-binding protein tau at sites that are hyperphosphorylated by GSK-3 and CDK5/p35 in Alzheimer's disease (cross-reacting with Alzheimer's-specific AT100 antibodies). CONCLUSIONS: The natural product hymenialdisine is a new kinase inhibitor with promising potential applications for treating neurodegenerative disorders.

Wnt factors in axonal remodelling and synaptogenesis.

Wiring' of the central nervous system is accomplished by the precise and co-ordinated behaviour of neuronal cells. Proper navigation of axons and formation of synaptic contacts with the correct targets are essential. Although several signalling molecules that control axon guidance, target selection and formation of synapses have been identified, little is known about how these proteins lead to changes in the axonal cytoskeleton. Wnt signalling factors have been shown to induce axonal remodelling in developing neurons. As several components of the Wnt signalling pathway are known, studies on Wnt factors could elucidate the mechanisms by which extracellular molecules regulate the neuronal cytoskeleton. Wnt-7a induces axonal spreading and subsequent increases in synaptic protein levels in mouse cerebellar neurons. These findings suggest a role for Wnt-7a in axon guidance and synapse formation in the developing cerebellum. Based on analyses of the axonal cytoskeleton, a model is proposed in which Wnt-7a induces axonal remodelling by inhibiting glycogen synthase kinase-3 beta (GSK-3 beta), a serine/threonine kinase. Inhibition of GSK-3 beta leads to a decrease in a phosphorylated form of microtubule-associated protein-1B (MAP-1B), a protein involved in microtubule assembly, and a concomitant decrease in the level of stable microtubules. This chapter discusses the novel role of Wnt factors in regulating the axonal cytoskeleton during neuronal development.

WNT signaling in the control of hair growth and structure.

Characterization of the molecular pathways controlling differentiation and proliferation in mammalian hair follicles is central to our understanding of the regulation of normal hair growth, the basis of hereditary hair loss diseases, and the origin of follicle-based tumors. We demonstrate that the proto-oncogene Wnt3, which encodes a secreted paracrine signaling molecule, is expressed in developing and mature hair follicles and that its overexpression in transgenic mouse skin causes a short-hair phenotype due to altered differentiation of hair shaft precursor cells, and cyclical balding resulting from hair shaft structural defects and associated with an abnormal profile of protein expression in the hair shaft. A putative effector molecule for WNT3 signaling, the cytoplasmic protein Dishevelled 2 (DVL2), is normally present at high levels in a subset of cells in the outer root sheath and in precursor cells of the hair shaft cortex and cuticle which lie immediately adjacent to Wnt3-expressing cells. Overexpression of Dvl2 in the outer root sheath mimics the short-hair phenotype produced by overexpression of Wnt3, supporting the hypothesis that Wnt3 and Dvl2 have the potential to act in the same pathway in the regulation of hair growth. These experiments demonstrate a previously unrecognized role for WNT signaling in the control of hair growth and structure, as well as presenting the first example of a mammalian phenotype resulting from overexpression of a Dvl gene and providing an accessible in vivo system for analysis of mammalian WNT signaling pathways.

Control of muscle fibre and motoneuron diversification.

Recent studies have elucidated both the mechanism of early formation of diverse muscle fibre types and the matching of diverse populations of motoneurons to their appropriate muscle targets. Highlights include the demonstration that distinct signals are necessary for the formation of several distinct myoblast populations in the vertebrate somite, the identification of motoneuron subtypes, studies of how motoneurons target appropriate muscles, and rapid progress on the Drosophila neuromuscular system. We propose a model in which four classes of decision control the patterning of both motoneurons and muscles.

Lithium and synaptic plasticity.

Lithium, a small cation, has been used in the treatment of bipolar disorders since its introduction in the 1950s by John Cade. Extensive research on the mechanism of action of lithium has revealed several possible targets. For some time, the most widely accepted action of lithium was its inhibitory effect on the synthesis of inositol, resulting in depletion of inositol with profound effects on neuronal signal transduction pathways. However, several studies show that some effects of lithium are not mediated through inositol depletion. Recent findings demonstrate that lithium directly inhibits, in a non-competitive fashion, the activity of glycogen synthase kinase (GSK)-3beta, a serine/threonine kinase highly expressed in the central nervous system. Interestingly, inhibition of GSK-3beta has been shown to regulate neuronal plasticity by inducing axonal remodelling and increasing the levels of synaptic proteins. These findings raise the possibility for developing new therapeutic approaches for the treatment of bipolar disorders.

Inhibition of GSK-3beta leading to the loss of phosphorylated MAP-1B is an early event in axonal remodelling induced by WNT-7a or lithium.

WNT-7a induces axonal spreading and branching in developing cerebellar granule neurons. This effect is mediated through the inhibition of GSK-3beta, a serine/threonine kinase and a component of the WNT pathway. Lithium, an inhibitor of GSK-3beta, mimics WNT-7a in granule cells. Here we examined further the effect of GSK-3beta inhibition on cytoskeletal re-organisation. Lithium induces axonal spreading and increases growth cone area and perimeter. This effect is associated with the absence or reduction of stable microtubules in spread areas. Lithium induces the loss of a phosphorylated form of MAP-1B, a microtubule associated protein involved in axonal outgrowth. Down-regulation of the phosphorylated MAP-1B, MAP-1B-P, from axonal processes occurs before axonal remodelling is evident. In vitro phosphorylation assays show that MAP-1B-P is generated by direct phosphorylation of MAP-1B by GSK-3beta. WNT-7a, like lithium, also leads to loss of MAP-1B-P from spread axons and growth cones. Our data suggest that WNT-7a and lithium induce changes in microtubule dynamics by inhibiting GSK-3beta which in turn lead to changes in the phosphorylation of MAP-1B. These findings suggest a novel role for GSK-3beta and WNTs in axonal remodelling and identify MAP-1B as a new target for GSK-3beta and WNT.

n beta geo, a combined selection and reporter gene for retroviral and transgenic studies.

Nuclear-targeted beta-galactosidase (beta-gal) is increasingly used as a genetic cell marker in vitro and in vivo. Nuclear sequestration concentrates beta-gal and permits sensitive identification of expressing cells and/or tissues without obscuring the cytoplasmic detail necessary for analysis of cell phenotype. Here, we report the construction and testing of a nuclear-targeted version of the beta geo fusion protein that combines nuclear localization with the ability to select expressing cells with the drug G418. This new marker gene functions efficiently in retroviral vectors and will be useful in identification and isolation of cells transfected in vitro and cells expressing transgenic or gene-targeted constructs in vivo.

Overexpression of the mouse dishevelled-1 protein inhibits GSK-3beta-mediated phosphorylation of tau in transfected mammalian cells.

Tau is a neuronal microtubule-associated protein whose function is modulated by phosphorylation. GSK-3beta is a tau kinase. GSK-3beta is part of the wingless signalling pathway and stimulation by wingless is predicted to down-regulate GSK-3beta activity. In Drosophila imaginal disc cells, overexpression of dishevelled, a component of the wingless pathway, mimics the wingless signal. We have therefore studied the effect that overexpression of the murine dishevelled-1 protein has on GSK-3beta-mediated phosphorylation of tau in transfected CHO cells. We find that co-transfection with dishevelled-1 is inhibitory to GSK-3beta-mediated tau phosphorylation. Tau is hyperphosphorylated in Alzheimer's disease and the possible relevance of these findings to Alzheimer's disease pathogenesis are discussed.

Expression of Tiam-1 in the developing brain suggests a role for the Tiam-1-Rac signaling pathway in cell migration and neurite outgrowth.

During development proper neuronal migration and neurite extension are essential for the formation of functional neuronal networks. These processes require the reorganization of the cytoskeleton by modifying the dynamics of actin filaments and microtubules. The Rho subfamily of GTPases regulates actin cytoskeletal changes during development. Tiam-1, a GDP-GTP exchange factor for the small GTPase Rac and implicated in tumor invasion and metastasis, is expressed in the developing CNS. To study the function of Tiam-1 in neuronal migration and neurite extension, we examined the pattern of Tiam-1 expression in weaver mice, in which cerebellar granule cells fail to migrate to their final position and subsequently die. Tiam-1 is expressed in wild-type granule cells as they migrate to the internal granular layer and send axone. In contrast, weaver homozygous animals do not express. Tiam-1 in premigratory granule cells. Heterozygous animals, in which granule cells exhibit a slow rate of migration, express low levels of Tiam-1. In the cerebral cortex, Tiam-1 is also expressed in migrating neurons. Our findings suggest that Tiam-1 contributes to cytoskeletal reorganization required during cell migration and neurite extension in defined neuronal populations, presumably by activation of Rac.

WNT-7a induces axonal remodeling and increases synapsin I levels in cerebellar neurons.

WNT factors play a key role in early patterning of the embryo. However, expression of Wnt genes after cell commitment suggests additional roles in later developmental processes. We report here that Wnt-7a is expressed in cerebellar granule cell neurons as they begin to extend processes and form synapses. WNT-7a increases axonal spreading and branching in cultured granule cells. Moreover, WNT-7a increases the levels of synapsin I, a presynaptic protein involved in synapse formation and function. Lithium mimics WNT-7a in granule cells by inhibiting GSK-3beta, a component of the WNT signaling pathway. These results suggest a direct effect of WNT-7a in the regulation of neuronal cytoskeleton and synapsin I in granule cell neurons. We propose that WNT proteins have a novel function in the formation of neuronal connections.

Regulation of angR, a gene with regulatory and biosynthetic functions in the pJM1 plasmid-mediated iron uptake system of Vibrio anguillarum.

AngR and the product(s) encoded in the trans-acting factor (TAF) region are necessary for the full expression of the pJM1 plasmid-mediated anguibactin iron-uptake system in Vibrio anguillarum (Va). In this report, we analyzed the factors that affect the expression of the angR gene. Northern blot analysis showed that angR encodes a 3.1-kb transcript which is expressed only under iron-limiting conditions. Measurement of steady-state RNA levels show that, under iron-limiting conditions, angR is positively regulated at the transcriptional level by product(s) of the Va TAF region. However, this enhancement of angR expression by TAF does not occur at high levels of the AngR protein, as assessed by using an angR::lacZ fusion in the presence of a construct containing angR under the control of ptac. We also report that repression of angR by iron could possibly be mediated by an endogenous Va antisense RNA beta, which contains a stem-loop structure complementary to the stem-loop structure located at the 5' end of angR.

Maintenance of Wnt-3 expression in Purkinje cells of the mouse cerebellum depends on interactions with granule cells.

Wnt genes encode secreted proteins implicated in cell fate changes during development. To define specific cell populations in which Wnt genes act, we have examined Wnt expression in the cerebellum. This part of the brain has a relatively simple structure and contains well-characterized cell populations. We found that Wnt-3 is expressed during development of the cerebellum and that expression is restricted to the Purkinje cell layer in the adult. Wnt-3 expression in Purkinje cells increases postnatally as granule cells start to make contacts with Purkinje cells. To investigate whether interactions with granule cells influence Wnt-3 expression in Purkinje cells, we examined gene expression in several mouse mutants, using the expression of En-2 to follow the fate of granule cells. In the weaver mutant, in which granule cells fail to migrate and subsequently die in the external granular layer, Wnt-3 expression was normal at postnatal day 15 (P15). At that time, some granule cells are still present in the external granular layer. At P28, however, when granule cells could no longer be detected, Wnt-3 expression was almost absent. In the meander tail mutant, in which the anterior cerebellar lobes lack granule cells, Wnt-3 expression was only detected in the normal posterior lobes. Since En genes are implicated in cell-cell interactions mediated by Wnt genes, we examined En-2/En-2 mutant mice, finding normal Wnt-3 expression, indicating that the effect of granule cells on the maintenance of Wnt-3 is not mediated by En-2. Our results show that Wnt-3 expression in Purkinje cells is modulated by their presynaptic granule cells at the time of neuronal maturation.

Regulation of the expression of bacterial iron transport genes: possible role of an antisense RNA as a repressor.

Expression of the iron transport gene, fatB and fatA, in the bacterium Vibrio anguillarum 775 is negatively regulated by the iron concentration in the medium. Here, we show that iron represses fatB and fatA mRNA levels and concomitantly induces the synthesis of an antisense RNA (RNA alpha). The presence of RNA alpha correlates with the inhibition of FatA protein synthesis and thus may play a role in the iron repression of fatA expression. Since the 5' end of RNA alpha maps 125 nucleotides upstream from the start codon of fatA and this RNA also extends into the coding region of fatB, it may also be involved in the iron regulation of fatB expression. RNA alpha may thus constitute a novel component of the bacterial iron regulatory circuit.

Regional expression of the Wnt-3 gene in the developing mouse forebrain in relationship to diencephalic neuromeres.

During early vertebrate development, a series of neuromeres divides the central nervous system from the forebrain to the spinal cord. Here we examine in more detail the expression of Wnt-3, a member of the Wnt gene family of secreted proteins, in the developing diencephalon, in comparison to the expression of the homeobox gene Dlx-1. In 9.5-day mouse embryos, Wnt-3 is expressed in a restricted area of the diencephalon before any morphological signs of subdivisions appear. Around embryonic day 11.5, Wnt-3 expression becomes restricted to one of the neuromeres of the diencephalon, the dorsal thalamus. Dlx-1 is expressed in a non-overlapping area immediately anterior to and abutting the Wnt-3 expressing domain, corresponding to the ventral thalamus. In addition, Wnt-3 is expressed in the midbrain-hindbrain region. In the adult mouse, Wnt-3 and Dlx-1 are expressed in subsets of neural cells derived from the original areas of expression in the diencephalon. Taken together, our results suggest that Wnt-3 and Dlx-1 provide positional information for the regional specification of neuromeres in the forebrain. The continued expression of these genes in the adult mouse brain suggests a distinct role in the mature CNS.

Regulation of the iron uptake system in Vibrio anguillarum: evidence for a cooperative effect between two transcriptional activators.

We have identified a 110-kDa polypeptide that has a trans-acting regulatory activity on the expression of the pJM1 plasmid iron-uptake genes in Vibrio anguillarum. This protein is encoded by the angR gene and maps in a 3.6-kilobase-pair pJM1 DNA region located downstream of the iron transport genes. Full expression of this gene occurs under iron-limiting conditions and requires a 2.9-kilobase-pair upstream region in cis that maps within the coding region of the OM2 outer membrane protein, essential for the transport of iron into the cell cytosol. Determination of the siderophore anguibactin levels as well as analysis of specific transcripts for anguibactin biosynthetic genes demonstrated that AngR and another transcriptional activator, Taf, regulate in a synergistic fashion the level of anguibactin production by activation of transcription of the anguibactin biosynthetic genes under iron-limiting conditions.

Increased production of the siderophore anguibactin mediated by pJM1-like plasmids in Vibrio anguillarum.

The virulence of the fish pathogen Vibrio anguillarum 775 is mediated by the pJM1 plasmid-specified iron uptake system which is expressed under conditions of iron limitation. Other V. anguillarum strains isolated from various geographical locations harbor plasmids that are highly related to pJM1 and that are also associated with the high-virulence phenotype of these strains. In this work, we found that a pJM1-like plasmid, pJHC1, from one of these virulent strains encoded an iron uptake system that resulted in an increased level of production of the siderophore anguibactin. The gene(s) responsible for increased anguibactin production was included within the iron uptake region of plasmid pJHC1. The cloned iron uptake regions of pJHC1 and pJM1 possessed identical restriction endonuclease maps, suggesting that the DNA region encoding those genes in pJHC1 may have diverged subtly from that in pJM1. Analysis of the iron uptake system from other V. anguillarum strains carrying pJM1-like plasmids demonstrated that strains originating from diseased fish from the Atlantic coast carry plasmids encoding an increased-siderophore-production phenotype, while strains isolated from Pacific Ocean locations behaved as the 775 strain.