A genome-wide survey of the evolutionarily conserved Wnt pathways in the sea urchin Strongylocentrotus purpuratus

The Wnt pathways are evolutionarily well-conserved signal transduction pathways that are known to play important roles in all Metazoans investigated to date. Here, we examine the Wnt pathway genes and target genes present in the genome of the echinoderm Strongylocentrotus purpuratus. Analysis of the Wnt genes revealed that eleven of the thirteen reported Wnt subfamilies are represented in sea urchin, with the intriguing identification of a Wnt-A ortholog thought to be absent in deuterostomes. A phylogenetic study of the Frizzled proteins, the Wnt receptors, performed throughout the animal kingdom showed that not all Frizzled subfamilies were present in the metazoan common ancestor, e.g. Fz3/6 emerged later during evolution. Using sequence analysis, orthologs of the vast majority of the cellular machinery involved in transducing the three types of Wnt pathways were found in the sea urchin genome. Furthermore, of about one hundred target genes identified in other organisms, more than half have clear echinoderm orthologs. Thus, these analyses produce new inputs in the evolutionary history of the Wnt genes in an animal occupying a position that offers great insights into the basal properties of deuterostomes.     

The Wnt/beta-catenin pathway regulates Gli-mediated Myf5 expression during somitogenesis

Canonical Wnt/beta-catenin signaling regulates the activation of the myogenic determination gene Myf5 at the onset of myogenesis, but the underlying molecular mechanism is unknown. Here, we report that the Wnt signal is transduced in muscle progenitor cells by at least two Frizzled (Fz) receptors (Fz1 and/or Fz6), through the canonical beta-catenin pathway, in the epaxial domain of newly formed somites. We show that Myf5 activation is dramatically reduced by blocking the Wnt/beta-catenin pathway in somite progenitor cells, whereas expression of activated beta-catenin is sufficient to activate Myf5 in somites but not in the presomitic mesoderm. In addition, we identified Tcf/Lef sequences immediately 5' to the Myf5 early epaxial enhancer. These sites determine the correct spatiotemporal expression of Myf5 in the epaxial domain of the somite, mediating the synergistic action of the Wnt/beta-catenin and the Shh/Gli pathways. Taken together, these results demonstrate that Myf5 is a direct target of Wnt/beta-catenin, and that its full activation requires a cooperative interaction between the canonical Wnt and the Shh/Gli pathways in muscle progenitor cells.     

Archenteron elongation in the sea urchin embryo is a microtubule-independent process

Earlier studies using colchicine (L. G. Tilney and J. R. Gibbins, 1969, J. Cell Sci. 5, 195-210) had suggested that intact microtubules (MTs) are necessary for archenteron elongation during the second phase of sea urchin gastrulation (secondary invagination), presumably by allowing secondary mesenchyme cells (SMCs) to extend their long filopodial processes. In light of subsequently discovered effects of colchicine on other cellular processes, the role of MTs in archenteron elongation in the sea urchin, Lytechinus pictus, has been reexamined. Immunofluorescent staining of ectodermal fragments and isolated archenterons reveals a characteristic pattern of MTs in the ectoderm and endoderm during gastrulation. Ectodermal cells exhibit arrays of MTs radiating away from the region of the basal body/ciliary rootlet and extending along the periphery of the cell, whereas endodermal cells exhibit a similar array of peripheral MTs emanating from the region of the apical ciliary rootlet facing the lumen of the archenteron. MTs are found primarily at the bases of the filopodia of normal SMCs. beta-Lumicolchicine (0.1 mM), an analog of colchicine which does not bind tubulin, inhibits secondary invagination, indicating that the effects previously ascribed to the disruption of MTs are probably due to the effects of colchicine on other cellular processes. The MT inhibitor nocodazole (5-10 micrograms/ml) added prior to secondary invagination does not prevent gastrulation or spontaneous exogastrulation, even though indirect immunofluorescence indicates that cytoplasmic MTs are completely disrupted in drug-treated embryos. Transverse tissue sections indicate that a comparable amount of cell rearrangement occurs in nocodazole-treated and control embryos. Significantly, SMCs in nocodazole-treated embryos often detach prematurely from the tip of the gut rudiment and extend abnormally large broad lamellipodial protrusions but are also capable of extending long slender filopodia comparable in length to those of control embryos. These results indicate that cytoplasmic MTs are not essential for either filopodial extension by SMCs or for the active epithelial cell rearrangement which accompanies elongation during sea urchin gastrulation.     

Wingless transduction by the Frizzled and Frizzled2 proteins of Drosophila.

Wingless (Wg) protein is a founding member of the Wnt family of secreted proteins which have profound organizing roles in animal development. Two members of the Frizzled (Fz) family of seven-pass transmembrane proteins, Drosophila Fz and Fz2, can bind Wg and are candidate Wg receptors. However, null mutations of the fz gene have little effect on Wg signal transduction and the lack of mutations in the fz2 gene has thus far prevented a rigorous examination of its role in vivo. Here we describe the isolation of an amber mutation of fz2 which truncates the coding sequence just after the amino-terminal extracellular domain and behaves genetically as a loss-of-function allele. Using this mutation, we show that Wg signal transduction is abolished in virtually all cells lacking both Fz and Fz2 activity in embryos as well as in the wing imaginal disc. We also show that Fz and Fz2 are functionally redundant: the presence of either protein is sufficient to confer Wg transducing activity on most or all cells throughout development. These results extend prior evidence of a ligand-receptor relationship between Wnt and Frizzled proteins and suggest that Fz and Fz2 are the primary receptors for Wg in Drosophila.     

Structural analysis of the interaction between Dishevelled2 and clathrin AP-2 adaptor, a critical step in noncanonical Wnt signaling

Wnt association with its receptor, Frizzled (Fz), and recruitment by the latter of an adaptor, Dishevelled (Dvl), initiates signaling through at least two distinct pathways ("canonical" and "noncanonical"). Endocytosis and compartmentalization help determine the signaling outcome. Our previous work has shown that Dvl2 links at least one Frizzled family member (Fz4) to clathrin-mediated endocytosis by interacting with the μ2 subunit of the AP-2 clathrin adaptor, through both a classical endocytic tyrosine motif and a so-called "DEP domain." We report here the crystal structure of a chimeric protein that mimics the Dvl2-μ2 complex. The DEP domain binds at one end of the elongated, C-terminal domain of μ2. This domain:domain interface shows that parts of the μ2 surface distinct from the tyrosine-motif site can help recruit specific receptors or adaptors into a clathrin coated pit. Mutation of residues at the DEP-μ2 contact or in the tyrosine motif reduce affinity of Dvl2 for μ2 and block efficient internalization of Fz4 in response to ligation by Wnt5a. The crystal structure has thus allowed us to identify the specific interaction that leads to Frizzled uptake and to downstream, noncanonical signaling events.     

Identification of a specific inhibitor of the dishevelled PDZ domain

The Wnt signaling pathways are involved in embryo development as well as in tumorigenesis. Dishevelled (Dvl) transduces Wnt signals from the receptor Frizzled (Fz) to downstream components in canonical and noncanonical Wnt signaling pathways. The Dvl PDZ domain is thought to play an essential role in both pathways, and we recently demonstrated that the Dvl PDZ domain binds directly to Fz receptors. In this study, using structure-based virtual ligand screening, we identified an organic molecule (NSC668036) from the National Cancer Institute small-molecule library that can bind to the Dvl PDZ domain. We then used molecular dynamics simulation to analyze the binding between the PDZ domain and NSC668036 in detail. In addition, we showed that, in Xenopus, as expected, NSC668036 inhibited the signaling induced by Wnt3A. This compound provides a basis for rational design of high-affinity inhibitors of the PDZ domain, which can block Wnt signaling by interrupting the Fz-Dvl interaction.     

Multiplicity of the interactions of Wnt proteins and their receptors

Wnts are secreted proteins that are essential for a wide array of developmental and physiological processes. They signal across the plasma membranes by interacting with serpentine receptors of the Frizzled (Fz) family and members of the low-density-lipoprotein receptor-related protein (LRP) family. Recent advances in the Wnt signaling field have revealed that Wnt-unrelated proteins activate or suppress Wnt signaling by binding to Fzs or LRP5/6 and that atypical receptor tyrosine kinases mediate Wnt signaling independently of Fz and/or function as a Fz co-receptor. This review highlights recent progress in our understanding of the multiplicity of Wnts and their receptors. We discuss how the interaction between the ligands and receptors activate distinct intracellular signaling pathways. We also discuss how intracellular trafficking of Wnt signaling components can regulate the sensitivity of cells to Wnts.     

Wnt signaling inhibits adipogenesis through beta-catenin-dependent and -independent mechanisms

Wnt signaling has been reported to block apoptosis and regulate differentiation of mesenchymal progenitors through inhibition of glycogen synthase kinase 3 and stabilization of beta-catenin. The effects of Wnt in preadipocytes may be mediated through Frizzled (Fz) 1 and/or Fz2 as these Wnt receptors are expressed in preadipocytes and their expression declines upon induction of differentiation. We ectopically expressed constitutively active chimeras between Wnt8 and Fz1 or Fz2 in preadipocytes and mesenchymal precursor cells. Our results indicated that activated Fz1 increases stability of beta-catenin, inhibits apoptosis, induces osteoblastogenesis, and inhibits adipogenesis. Although activated Fz2 does not influence apoptosis or osteoblastogenesis, it inhibits adipogenesis through a mechanism independent of beta-catenin. An important mediator of the beta-catenin-independent pathway appears to be calcineurin because inhibitors of this serine/threonine phosphatase partially rescue the block to adipogenesis caused by Wnt3a or activated Fz2. These data supported a model in which Wnt signaling inhibits adipogenesis through both beta-catenin-dependent and beta-catenin-independent mechanisms.     

The role of secondary mesenchyme cells during sea urchin gastrulation studied by laser ablation

It has long been thought that traction exerted by filopodia of secondary mesenchyme cells (SMCs) is a sufficient mechanism to account for elongation of the archenteron during sea urchin gastrulation. The filopodial traction hypothesis has been directly tested here by laser ablation of SMCs in gastrulae of the sea urchin, Lytechinus pictus. When SMCs are ablated at the onset of secondary invagination, the archenteron doubles in length at the normal rate of elongation, but advance of the tip of the archenteron stops at the 2/3 gastrula stage. In contrast, when all SMCs are ablated at or following the 2/3 gastrula stage, further elongation does not occur. However, if a few SMCs are allowed to remain in 2/3-3/4 gastrulae, elongation continues, although more slowly than in controls. The final length of archenterons in embryos ablated at the 1/3-1/2 gastrula stage is virtually identical to the final length of everted archenterons in LiCl-induced exogastrulae; since filopodial traction is not exerted in either case, an alternate, common mechanism of elongation probably operates in both cases. These results suggest that archenteron elongation involves two processes: (1) active, filopodia-independent elongation, which depends on active cell rearrangement and (2) filopodia-dependent elongation, which depends on mechanical tension exerted by the filopodia.     

The dynamic expression pattern of frzb-1 suggests multiple roles in chick development

The Wnt family of secreted proteins has been shown to have multiple roles in embryonic development. Wnt signals are thought to be propagated by binding to the cysteine-rich extracellular domain (CRD) of Frizzled, a seven-transmembrane-domain cell surface receptor. Secreted Frizzled-related proteins (generally denoted Frzb or Sfrp) possess a domain with a high degree of sequence identity and structural similarity with the CRD of Frizzled. Current data indicate that the cysteine-rich domain of secreted Frzb proteins can bind Wnt proteins, suggesting the possibility that Frzbs compete with membrane-bound Frizzled for Wnt binding and consequently act as competitive inhibitors of Wnt signaling. In order to gain a better understanding of the potential roles of Frzb-1 in chick development, we utilized the polymerase chain reaction to isolate a partial cDNA of the chick orthologue of frzb-1, cfrzb-1, and compared its expression pattern to that of Wnt-1, Wnt-3a, Wnt-5a, Wnt-7a, and Wnt-8c. Whole-mount in situ hybridizations have revealed three major phases of expression for cfrzb-1 in the developing chick. The earliest expression of cfrzb-1 is in cells fated to become neural ectoderm in streak-stage embryos. Expression of cfrzb-1 in the neural ectoderm continues up through stage 8. After stage 8, cfrzb-1 expression is gradually attenuated in the closing neural tube of the trunk and is concomitantly up-regulated in neural crest cells. Finally, cfrzb-1 appears in the condensing mesenchyme of the bones in both the limb and the trunk in stage 25+ embryos. Comparative analysis of the cfrzb-1 and the Wnt gene expression patterns suggests possible interactions between cFrzb-1 and all of the Wnt family members examined.     

Developmental characterization of the gene for laminin alpha-chain in sea urchin embryos.

We describe the isolation and characterization of a cDNA clone encoding a region of the carboxy terminal globular domain (G domain) of the alpha-1 chain of laminin from the sea urchin, Strongylocentrotus purpuratus. Sequence analysis indicates that the 1.3 kb cDNA (spLAM-alpha) encodes the complete G2 and G3 subdomains of sea urchin a-laminin. The 11 kb spLAM-alpha mRNA is present in the egg and declines slightly in abundance during development to the pluteus larva. The spLAM-alpha gene is also expressed in a variety of adult tissues. Whole mount in situ hybridization of gastrula stage embryos indicates that ectodermal and endodermal epithelia and mesenchyme cells contain the spLAM-alpha mRNA. Immunoprecipitation experiments using an antibody made to a recombinant fusion protein indicates spLAM-alpha protein is synthesized continuously from fertilization as a 420 kDa protein which accumulates from low levels in the egg to elevated levels in the pluteus larva. Light and electron microscopy identify spLAM-alpha as a component of the basal lamina. Blastocoelic microinjection of an antibody to recombinant spLAM-alpha perturbs gastrulation and skeleton formation by primary mesenchyme cells suggesting an important role for laminin in endodermal and mesodermal morphogenesis.     

Wnt5a regulates distinct signalling pathways by binding to Frizzled2

Wnt5a regulates multiple intracellular signalling cascades, but how Wnt5a determines the specificity of these pathways is not well understood. This study examined whether the internalization of Wnt receptors affects the ability of Wnt5a to regulate its signalling pathways. Wnt5a activated Rac in the β‐catenin‐independent pathway, and Frizzled2 (Fz2) and Ror1 or Ror2 were required for this action. Fz2 was internalized through a clathrin‐mediated route in response to Wnt5a, and inhibition of clathrin‐dependent internalization suppressed the ability of Wnt5a to activate Rac. As another action of Wnt5a, it inhibited Wnt3a‐dependent lipoprotein receptor‐related protein 6 (LRP6) phosphorylation and β‐catenin accumulation. Wnt3a‐dependent phosphorylation of LRP6 was enhanced in Wnt5a knockout embryonic fibroblasts. Fz2 was also required for the Wnt3a‐dependent accumulation of β‐catenin, and Wnt5a competed with Wnt3a for binding to Fz2 in vitro and in intact cells, thereby inhibiting the β‐catenin pathway. This inhibitory action of Wnt5a was not affected by the impairment of clathrin‐dependent internalization. These results suggest that Wnt5a regulates distinct pathways through receptor internalization‐dependent and ‐independent mechanisms.     

Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation of endoderm and mesodermal cell lineages

The entry of beta-catenin into vegetal cell nuclei beginning at the 16-cell stage is one of the earliest known molecular asymmetries seen along the animal-vegetal axis in the sea urchin embryo. Nuclear beta-catenin activates a vegetal signaling cascade that mediates micromere specification and specification of the endomesoderm in the remaining cells of the vegetal half of the embryo. Only a few potential target genes of nuclear beta-catenin have been functionally analyzed in the sea urchin embryo. Here, we show that SpWnt8, a Wnt8 homolog from Strongylocentrotus purpuratus, is zygotically activated specifically in 16-cell-stage micromeres in a nuclear beta-catenin-dependent manner, and its expression remains restricted to the micromeres until the 60-cell stage. At the late 60-cell stage nuclear beta-catenin-dependent SpWnt8 expression expands to the veg2 cell tier. SpWnt8 is the only signaling molecule thus far identified with expression localized to the 16-60-cell stage micromeres and the veg2 tier. Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. These results establish SpWnt8 as a critical component of the endomesoderm regulatory network in the sea urchin embryo.     

A switch in the cellular basis of skeletogenesis in late-stage sea urchin larvae

Primary mesenchyme cells (PMCs) are solely responsible for the skeletogenesis during early larval development of the sea urchin, but the cells responsible for late larval and adult skeletal formation are not clear. To investigate the origin of larval and adult skeletogenic cells, I first performed transplantation experiments in Pseudocentrotus depressus and Hemicentrotus pulcherrimus, which have different skeletal phenotypes. When P. depressus PMCs were transplanted into H. pulcherrimus embryos, the donor phenotype was observed only in the early larval stage, whereas when secondary mesenchyme cells (SMCs) were transplanted, the donor phenotype was observed in late and metamorphic larvae. Second, a reporter construct driven by the spicule matrix protein 50 (SM50) promoter was introduced into fertilized eggs and their PMCs/SMCs were transplanted. In the resultant 6-armed pluteus, green fluorescent protein (GFP) expression was observed in both PMC and SMC transplantations, suggesting SMC participation in late skeletogenesis. Third, transplanted PMCs or SMCs tagged with GFP were analyzed by PCR in the transgenic chimeras. As a result, SMCs were detected in both larval and adult stages, but GFP from PMCs was undetectable after metamorphosis. Thus, it appears that SMCs participate in skeletogenesis in late development and that PMCs disappear in the adult sea urchin, suggesting that the skeletogenesis may pass from PMCs to SMCs during the late larval stage.     

Reconstitution of a Frizzled8��Wnt3a��LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6

Wnt/β-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/β-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD·Wnt3a·LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth β-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/β-catenin signaling by Dkk1.     

Subcellular localization of frizzled receptors, mediated by their cytoplasmic tails, regulates signaling pathway specificity

The Frizzled (Fz; called here Fz1) and Fz2 receptors have distinct signaling specificities activating either the canonical Wnt/β-catenin pathway or Fz/planar cell polarity (PCP) signaling in Drosophila. The regulation of signaling specificity remains largely obscure. We show that Fz1 and Fz2 have different subcellular localizations in imaginal disc epithelia, with Fz1 localizing preferentially to apical junctional complexes, and Fz2 being evenly distributed basolaterally. The subcellular localization difference directly contributes to the signaling specificity outcome. Whereas apical localization favors Fz/PCP signaling, it interferes with canonical Wnt/β-catenin signaling. Receptor localization is mediated by sequences in the cytoplasmic tail of Fz2 that appear to block apical accumulation. Based on these data, we propose that subcellular Fz localization, through the association with other membrane proteins, is a critical aspect in regulating the signaling specificity within the Wnt/Fz signaling pathways.     

Amyloid-beta binds to the extracellular cysteine-rich domain of Frizzled and inhibits Wnt/beta-catenin signaling

The amyloid-beta peptide (Abeta) plays a major role in neuronal dysfunction and neurotoxicity in Alzheimer disease. However, the signal transduction mechanisms involved in Abeta-induced neuronal dysfunction remain to be fully elucidated. A major current unknown is the identity of the protein receptor(s) involved in neuronal Abeta binding. Using phage display of peptide libraries, we have identified a number of peptides that bind Abeta and are homologous to neuronal receptors putatively involved in Abeta interactions. We report here on a cysteine-linked cyclic heptapeptide (denominated cSP5) that binds Abeta with high affinity and is homologous to the extracellular cysteine-rich domain of several members of the Frizzled (Fz) family of Wnt receptors. Based on this homology, we investigated the interaction between Abeta and Fz. The results show that Abeta binds to the Fz cysteine-rich domain at or in close proximity to the Wnt-binding site and inhibits the canonical Wnt signaling pathway. Interestingly, the cSP5 peptide completely blocks Abeta binding to Fz and prevents inhibition of Wnt signaling. These results indicate that the Abeta-binding site in Fz is homologous to cSP5 and that this is a relevant target for Abeta-instigated neurotoxicity. Furthermore, they suggest that blocking the interaction of Abeta with Fz might lead to novel therapeutic approaches to prevent neuronal dysfunction in Alzheimer disease.     

Direct binding of the PDZ domain of Dishevelled to a conserved internal sequence in the C-terminal region of Frizzled

The cytoplasmic protein Dishevelled (Dvl) and the associated membrane-bound receptor Frizzled (Fz) are essential in canonical and noncanonical Wnt signaling pathways. However, the molecular mechanisms underlying this signaling are not well understood. By using NMR spectroscopy, we determined that an internal sequence of Fz binds to the conventional peptide binding site in the PDZ domain of Dvl; this type of site typically binds to C-terminal binding motifs. The C-terminal region of the Dvl inhibitor Dapper (Dpr) and Frodo bound to the same site. In Xenopus, Dvl binding peptides of Fz and Dpr/Frodo inhibited canonical Wnt signaling and blocked Wnt-induced secondary axis formation in a dose-dependent manner, but did not block noncanonical Wnt signaling mediated by the DEP domain. Together, our results identify a missing molecular connection within the Wnt pathway. Differences in the binding affinity of the Dvl PDZ domain and its binding partners may be important in regulating signal transduction by Dvl.