Wnt信号转导及其生物效应

 Wnt蛋白与其下游效应物构成一组重要的信号转导通路 .信号转导过程包括 :Wnt首先激活细胞表面受体佛力子 (FZ) ,活化的FZ将通过Dvl、CKIε抑制糖原合成酶激酶 3β ,继而拮抗 β 链结素( β cat)催毁器的作用 ,使胞浆中 β cat积聚并进入核内 .β cat在核内与转录因子LEF TCF协作 ,激活控制胚胎发育和细胞命运的靶基因 ;活化的FZ还经激活JNK及Flamingo来影响细胞骨架的聚合 ,以决定细胞的平面极性及纺缍体定位     

Subcellular localization and signaling properties of dishevelled in developing vertebrate embryos

The Dishevelled protein mediates several diverse biological processes. Intriguingly, within the same tissues where Xenopus Dishevelled (Xdsh) controls cell fate via canonical Wnt signaling, it also controls cell polarity via the vertebrate planar cell polarity (PCP) cascade [1, 2, 3, 4, 5, 6, 7, 8 and 9]. The relationship between subcellular localization of Dishevelled and its signaling activities remains unclear; conflicting results have been reported depending upon the organism and cell types examined [8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20]. We have approached this issue by developing new reagents to sequester wild-type Dishevelled protein either at the cell membrane or away from the cell membrane. Removal of Dishevelled from the cell membrane disrupts convergent extension by preventing Rho/Rac activation and mediolateral cell polarization. By manipulating the subcellular localization of K-->M (dsh1), we show that this mutation inhibits Dishevelled activation of Rac, regardless of its subcellular localization. These data demonstrate that membrane localization of Dishevelled is a prerequisite for vertebrate PCP signaling. However, both membrane-targeted and cytoplasm-targeted Dishevelled can potently activate canonical Wnt signaling, suggesting that local concentration of Dishevelled protein, but not its spatial localization, is central to canonical Wnt signaling. These results suggest that in vertebrate embryos, subcellular localization is insufficient to account for the pathway specificity of Dishevelled in the canonical Wnt versus PCP signaling cascades.     

Wnt signaling positions neuromuscular connectivity by inhibiting synapse formation in C. elegans

Nervous system function is mediated by a precisely patterned network of synaptic connections. While several cell-adhesion and secreted molecules promote the assembly of synapses, the contribution of signals that negatively regulate synaptogenesis is not well understood. We examined synapse formation in the Caenorhabditis elegans motor neuron DA9, whose presynapses are restricted to a specific segment of its axon. We report that the Wnt lin-44 localizes the Wnt receptor lin-17/Frizzled (Fz) to a subdomain of the DA9 axon that is devoid of presynaptic specializations. When this signaling pathway, composed of the Wnts lin-44 and egl-20, lin-17/Frizzled and dsh-1/Dishevelled, is compromised, synapses develop ectopically in this subdomain. Conversely, overexpression of LIN-44 in cells adjacent to DA9 is sufficient to expand LIN-17 localization within the DA9 axon, thereby inhibiting presynaptic assembly. These results suggest that morphogenetic signals can spatially regulate the patterning of synaptic connections by subdividing an axon into discrete domains.     

Synaptic specificity is generated by the synaptic guidepost protein SYG-2 and its receptor, SYG-1

Synaptic connections in the nervous system are directed onto specific cellular and subcellular targets. Synaptic guidepost cells in the C. elegans vulval epithelium drive synapses from the HSNL motor neuron onto adjacent target neurons and muscles. Here, we show that the transmembrane immunoglobulin superfamily protein SYG-2 is a central component of the synaptic guidepost signal. SYG-2 is expressed transiently by primary vulval epithelial cells during synapse formation. SYG-2 binds SYG-1, the receptor on HSNL, and directs SYG-1 accumulation and synapse formation to adjacent regions of HSNL. syg-1 and syg-2 mutants have defects in synaptic specificity; the HSNL neuron forms fewer synapses onto its normal targets and forms ectopic synapses onto inappropriate targets. Misexpression of SYG-2 in secondary epithelial cells causes aberrant accumulation of SYG-1 and synaptic markers in HSNL adjacent to the SYG-2-expressing cells. Our results indicate that local interactions between immunoglobulin superfamily proteins can determine specificity during synapse formation.     

Functional dissection of drosophila capricious: its novel roles in neuronal pathfinding and selective synapse formation

Drosophila Capricious (CAPS) is a transmembrane protein with leucine-rich repeat (LRR) motifs, expressed on small subsets of neurons and muscles, including muscle 12 and the motoneurons that innervate it (muscle 12 MNs). Panmuscle ectopic expression of CAPS alters the target specificity of muscle 12 MNs, indicating that CAPS can function in muscles as a target recognition molecule. In this study, we first examined the effect of ectopic panneural expression of CAPS on the motoneuronal circuit. We found that panneural expression of CAPS alters the pathfinding of muscle 12 MNs. The defect appeared to be caused by changes in the steering behavior of muscle 12 MNs at a specific choice point along their pathway to the target muscle. These results revealed a novel function of CAPS in axon pathfinding. We then performed deletion analyses of CAPS. We expressed CAPS lacking the intracellular domain in all neurons or in all muscles, and studied their ability to induce the pathfinding and targeting phenotypes. We found that the function of muscularly expressed CAPS in target recognition is intracellular domain dependent, whereas the function of neurally expressed CAPS in pathfinding is not, suggesting that CAPS may function in neurons and muscles in a different manner. The requirement of the intracellular domain for the function of muscularly expressed CAPS suggests the presence of a signaling event within muscle cells that is essential for selective synapse formation.     

Forked end: a novel transmembrane protein involved in neuromuscular specificity in drosophila identified by gain-of-function screening

The Drosophila neuromuscular connectivity provides an excellent model system for studies on target recognition and selective synapse formation. To identify molecules involved in neuromuscular recognition, we conducted gain-of-function screening for genes whose forced expression in all muscles alters the target specificity. We report here the identification of a novel transmembrane protein, Forked end (FEND), encoded by the fend gene, by the said screening. When the FEND expression was induced in all muscles, motoneurons that normally innervate muscle 12 formed ectopic synapses on a neighboring muscle 13. The target specificity of these motoneurons was also altered in the loss-of-function mutant of fend. During embryonic development, fend mRNA was detected in a subset of cells in the central nervous system and in the periphery. These results suggest that FEND is a novel axon guidance molecule involved in neuromuscular specificity.     

A Wnt-Frz/Ror-Dsh Pathway Regulates Neurite Outgrowth in Caenorhabditis elegans

One of the challenges to understand the organization of the nervous system has been to determine how axon guidance molecules govern axon outgrowth. Through an unbiased genetic screen, we identified a conserved Wnt pathway which is crucial for anterior-posterior (A/P) outgrowth of neurites from RME head motor neurons in Caenorhabditis elegans. The pathway is composed of the Wnt ligand CWN-2, the Frizzled receptors CFZ-2 and MIG-1, the co-receptor CAM-1/Ror, and the downstream component Dishevelled/DSH-1. Among these, CWN-2 acts as a local attractive cue for neurite outgrowth, and its activity can be partially substituted with other Wnts, suggesting that spatial distribution plays a role in the functional specificity of Wnts. As a co-receptor, CAM-1 functions cell-autonomously in neurons and, together with CFZ-2 and MIG-1, transmits the Wnt signal to downstream effectors. Yeast two-hybrid screening identified DSH-1 as a binding partner for CAM-1, indicating that CAM-1 could facilitate CWN-2/Wnt signaling by its physical association with DSH-1. Our study reveals an important role of a Wnt-Frz/Ror-Dsh pathway in regulating neurite A/P outgrowth.     

Association of Dishevelled with the clathrin AP-2 adaptor is required for Frizzled endocytosis and planar cell polarity signaling

Upon activation by Wnt, the Frizzled receptor is internalized in a process that requires the recruitment of Dishevelled. We describe a novel interaction between Dishevelled2 (Dvl2) and micro2-adaptin, a subunit of the clathrin adaptor AP-2; this interaction is required to engage activated Frizzled4 with the endocytic machinery and for its internalization. The interaction of Dvl2 with AP-2 requires simultaneous association of the DEP domain and a peptide YHEL motif within Dvl2 with the C terminus of micro2. Dvl2 mutants in the YHEL motif fail to associate with micro2 and AP-2, and prevent Frizzled4 internalization. Corresponding Xenopus Dishevelled mutants show compromised ability to interfere with gastrulation mediated by the planar cell polarity (PCP) pathway. Conversely, a Dvl2 mutant in its DEP domain impaired in PCP signaling exhibits defective AP-2 interaction and prevents the internalization of Frizzled4. We suggest that the direct interaction of Dvl2 with AP-2 is important for Frizzled internalization and Frizzled/PCP signaling.     

β-Catenin stabilization in skeletal muscles, but not in motor neurons, leads to aberrant motor innervation of the muscle during neuromuscular development in mice

β-Catenin, a key component of the Wnt signaling pathway, has been implicated in the development of the neuromuscular junction (NMJ) in mice, but its precise role in this process remains unclear. Here we use a β-catenin gain-of-function mouse model to stabilize β-catenin selectively in either skeletal muscles or motor neurons. We found that β-catenin stabilization in skeletal muscles resulted in increased motor axon number and excessive intramuscular nerve defasciculation and branching. In contrast, β-catenin stabilization in motor neurons had no adverse effect on motor innervation pattern. Furthermore, stabilization of β-catenin, either in skeletal muscles or in motor neurons, had no adverse effect on the formation and function of the NMJ. Our findings demonstrate that β-catenin levels in developing muscles in mice are crucial for proper muscle innervation, rather than specifically affecting synapse formation at the NMJ, and that the regulation of muscle innervation by β-catenin is mediated by a non-cell autonomous mechanism.     

R-spondin1 is a high affinity ligand for LRP6 and induces LRP6 phosphorylation and beta-catenin signaling

R-spondin proteins are newly identified secreted molecules that activate beta-catenin signaling. However, the mechanism of R-spondin action and its relationship with Wnt signaling remain unclear. Here we show that human R-spondin1 (hRspo1) is a high affinity ligand for the Wnt co-receptor LRP6 (K(d) = 1.2 nm). hRspo1 induces glycogen synthase kinase 3-dependent phosphorylation and activation of LRP6. DKK1, an LRP6 antagonist, inhibits hRspo1-induced LRP6 phosphorylation. We further demonstrate that hRspo1 synergizes with Frizzled5 in Xenopus axis induction assays and induces the phosphorylation of Dishevelled, a cytoplasmic component downstream of Frizzled function. Our study reveals interesting similarity and distinction between Wnt and R-spondin signaling.     

Behavioral and synaptic defects in C. elegans lacking the NK-2 homeobox gene ceh-28

C. elegans pharyngeal behavior consists of two distinct types of muscle contractions, termed pumping and peristalsis. Pumping ingests and concentrates bacteria in the anterior pharyngeal lumen, and it is occasionally followed by a transient peristaltic contraction that carries ingested bacteria through the posterior pharyngeal isthmus. These behaviors are controlled by a small pharyngeal nervous system consisting of 20 neurons that is almost completely independent of the extra-pharyngeal nervous system. The cholinergic motor neuron M4 controls peristalsis via synapses with the posterior isthmus muscles. Here we show that the NK-2 family homeobox gene ceh-28 is expressed in M4, where it regulates synapse assembly and peristalsis. ceh-28 mutants exhibit frequent and prolonged peristalses, and treatment with agonists or antagonists of muscarinic acetylcholine receptors can phenocopy or suppress ceh-28 mutant defects, respectively. Synapses in ceh-28 mutant M4 cells are irregularly spaced and sized, and they are abnormally located along the full length of the isthmus. We suggest that CEH-28 inhibits synaptogenesis, and that ceh-28 mutant behavioral defects result from excessive or ectopic stimulation of muscarinic acetylcholine receptors in the isthmus muscles.     

Dimerization choices control the ability of axin and dishevelled to activate c-Jun N-terminal kinase/stress-activated protein kinase

Axin and Dishevelled are two downstream components of the Wnt signaling pathway. Dishevelled is a positive regulator and is placed genetically between Frizzled and glycogen synthase kinase-3beta, whereas Axin is a negative regulator that acts downstream of glycogen synthase kinase-3beta. It is intriguing that they each can activate the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) when expressed in the cell. We set out to address if Axin and Dishevelled are functionally cooperative, antagonistic, or entirely independent, in terms of the JNK activation event. We found that in contrast to Axin, Dvl2 activation of JNK does not require MEKK1, and complex formation between Dvl2 and Axin is independent of Axin-MEKK1 binding. Furthermore, Dvl2-DIX and Dvl2-DeltaDEP proteins deficient for JNK activation can attenuate Axin-activated JNK activity by disrupting Axin dimerization. However, Axin-DeltaMID, Axin-DeltaC, and Axin-CT proteins deficient for JNK activation cannot interfere with Dvl2-activated JNK activity. These results indicate that unlike the strict requirement of homodimerization for Axin function, Dvl2 can activate JNK either as a monomer or homodimer/heterodimer. We suggest that there may be a switch mechanism based on dimerization combinations, that commands cells to activate Wnt signaling or JNK activation, and to turn on specific activators of JNK in response to various environmental cues.     

Wnt/PCP proteins regulate stereotyped axon branch extension in Drosophila

Branching morphology is a hallmark feature of axons and dendrites and is essential for neuronal connectivity. To understand how this develops, I analyzed the stereotyped pattern of Drosophila mushroom body (MB) neurons, which have single axons branches that extend dorsally and medially. I found that components of the Wnt/Planar Cell Polarity (PCP) pathway control MB axon branching. frizzled mutant animals showed a predominant loss of dorsal branch extension, whereas strabismus (also known as Van Gogh) mutants preferentially lost medial branches. Further results suggest that Frizzled and Strabismus act independently. Nonetheless, branching fates are determined by complex Wnt/PCP interactions, including interactions with Dishevelled and Prickle that function in a context-dependent manner. Branching decisions are MB-autonomous but non-cell-autonomous as mutant and non-mutant neurons regulate these decisions collectively. I found that Wnt/PCP components do not need to be asymmetrically localized to distinct branches to execute branching functions. However, Prickle axonal localization depends on Frizzled and Strabismus.     

Prickle 1 regulates cell movements during gastrulation and neuronal migration in zebrafish

During vertebrate gastrulation, mesodermal and ectodermal cells undergo convergent extension, a process characterised by prominent cellular rearrangements in which polarised cells intercalate along the medio-lateral axis leading to elongation of the antero-posterior axis. Recently, it has become evident that a noncanonical Wnt/Frizzled (Fz)/Dishevelled (Dsh) signalling pathway, which is related to the planar-cell-polarity (PCP) pathway in flies, regulates convergent extension during vertebrate gastrulation. Here we isolate and functionally characterise a zebrafish homologue of Drosophila prickle (pk), a gene that is implicated in the regulation of PCP. Zebrafish pk1 is expressed maternally and in moving mesodermal precursors. Abrogation of Pk1 function by morpholino oligonucleotides leads to defective convergent extension movements, enhances the silberblick (slb)/wnt11 and pipetail (Ppt)/wnt5 phenotypes and suppresses the ability of Wnt11 to rescue the slb phenotype. Gain-of-function of Pk1 also inhibits convergent extension movements and enhances the slb phenotype, most likely caused by the ability of Pk1 to block the Fz7-dependent membrane localisation of Dsh by downregulating levels of Dsh protein. Furthermore, we show that pk1 interacts genetically with trilobite (tri)/strabismus to mediate the caudally directed migration of cranial motor neurons and convergent extension. These results indicate that, during zebrafish gastrulation Pk1 acts, in part, through interaction with the noncanonical Wnt11/Wnt5 pathway to regulate convergent extension cell movements, but is unlikely to simply be a linear component of this pathway. In addition, Pk1 interacts with Tri to mediate posterior migration of branchiomotor neurons, probably independent of the noncanonical Wnt pathway.     

Dishevelled: at the crossroads of divergent intracellular signaling pathways.

During the development of multicellular organisms the formation of complex patterns relies on specific cell-cell signaling events. For tissues to become spatially organized and cells to become committed to specialized fates it is absolutely crucial for proper development that the underlying signaling systems receive and route information correctly. Recently, a wealth of genetic and biochemical experimental data has been collected about prevalent evolutionary conserved signaling families, such as the Wnts, Dpp/BMPs, and Hedgehogs, in flies, worms, and vertebrates. Paradoxically, members of a particular signaling family often have receptors with similar biochemical binding properties, though they activate different intracellular pathways in vivo and can be phenotypically distinguished. How are their specific biological responses then generated? With respect to signaling specificity in Wnt pathways, Dishevelled is an intriguing protein; in Drosophila melanogaster it is required in two distinct signaling pathways, that share Frizzled receptors of similar structure, but have distinct intracellular signaling routes. Recent results suggest that Dishevelled is a multifunctional protein at the crossroads of divergent Wnt/Fz pathways. Dishevelled appears to be a key factor in Wnt signaling to read' signals coming from the plasma membrane and route them into the correct intracellular pathways.     

Forked end: a novel transmembrane protein involved in neuromuscular specificity in drosophila identified by gain‐of‐function screening

The Drosophila neuromuscular connectivity provides an excellent model system for studies on target recognition and selective synapse formation. To identify molecules involved in neuromuscular recognition, we conducted gain‐of‐function screening for genes whose forced expression in all muscles alters the target specificity. We report here the identification of a novel transmembrane protein, Forked end (FEND), encoded by the fend gene, by the said screening. When the FEND expression was induced in all muscles, motoneurons that normally innervate muscle 12 formed ectopic synapses on a neighboring muscle 13. The target specificity of these motoneurons was also altered in the loss‐of‐function mutant of fend. During embryonic development, fend mRNA was detected in a subset of cells in the central nervous system and in the periphery. These results suggest that FEND is a novel axon guidance molecule involved in neuromuscular specificity. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 205–214, 2002     

Dishevelled Proteins Are Associated with Olfactory Sensory Neuron Presynaptic Terminals

Olfactory sensory neurons (OSNs) project their axons from the olfactory epithelium toward the olfactory bulb (OB) in a heterogeneous and unsorted arrangement. However, as the axons approach the glomerular layer of the OB, axons from OSNs expressing the same odorant receptor (OR) sort and converge to form molecularly homogeneous glomeruli. Axon guidance cues, cell adhesion molecules, and OR induced activity have been implicated in the final targeting of OSN axons to specific glomeruli. Less understood, and often controversial, are the mechanisms used by OSN axons to initially navigate from the OE toward the OB. We previously demonstrated a role for Wnt and Frizzled (Fz) molecules in OSN axon extension and organization within the olfactory nerve. Building on that we now turned our attention to the downstream signaling cascades from Wnt-Fz interactions. Dishevelled (Dvl) is a key molecule downstream of Fz receptors. Three isoforms of Dvl with specific as well as overlapping functions are found in mammals. Here, we show that Dvl-1 expression is restricted to OSNs in the dorsal recess of the nasal cavity, and labels a unique subpopulation of glomeruli. Dvl-2 and Dvl-3 have a widespread distribution in both the OE and OB. Both Dvl-1 and Dvl-2 are associated with intra-glomerular pre-synaptic OSN terminals, suggesting a role in synapse formation/stabilization. Moreover, because Dvl proteins were observed in all OSN axons, we hypothesize that they are important determinants of OSN cell differentiation and axon extension.