A novel member of the low density lipoprotein receptor (LDLR) gene family has been identified and characterized. This gene, termed LDL receptor-related protein 6 (LRP6), encodes a transmembrane protein that has 71% identity and is structurally similar to the protein encoded by LRP5, a proposed candidate gene for type 1 diabetes located on human chromosome 11q13. LRP6 maps to human chromosome 12p11-p13. Mouse Lrp6 encodes a protein that has 98% identity to human LRP6 and maps to chromosome 6. Unlike other members of the LDLR family, LRP6 and LRP5 display a unique pattern of four epidermal growth factor (EGF) and three LDLR repeats in the extracellular domain. The cytoplasmic domain of LRP6 is not similar to other members of the LDLR family, while comparison with LRP5 reveals proline-rich motifs that may mediate protein-protein interactions. Thus, it is likely that LRP6 and LRP5 comprise a new class of the LDLR family (Brown, 1998).
A gene encoding a novel transmembrane protein has been identified by DNA sequence analysis within the insulin-dependent diabetes mellitus (IDDM) locus IDDM4 on chromosome 11q13. Based on its chromosomal position, this gene is a candidate for conferring susceptibility to diabetes. The gene, termed low-density lipoprotein receptor related protein 5 (LRP5), encodes a protein of 1615 amino acids that contains conserved modules which are characteristic of the low-density lipoprotein (LDL) receptor family. These modules include a putative signal peptide for protein export, four epidermal growth factor (EGF) repeats with associated spacer domains, three LDL-receptor (LDLR) repeats, a single transmembrane spanning domain, and a cytoplasmic domain. The encoded protein has a unique organization of EGF and LDLR repeats; therefore, LRP5 likely represents a new category of the LDLR family. Both human and mouse LRP5 cDNAs have been isolated and the encoded mature proteins are 95% identical, indicating a high degree of evolutionary conservation (Hey, 1998).
The isolation and characterization of rabbit and human cDNAs reveals a new low density lipoprotein receptor (LDLR)-related protein (LRP) designated as LRP5. Human LRP5 cDNA encodes a 1,616-amino acid type I membrane-like protein with three ligand binding repeats in its extracellular region. LDLR-deficient cells transduced by recombinant adenovirus containing human LRP5 exhibit increased binding of apolipoprotein E (apoE)-enriched beta-migrating very low density lipoprotein. Northern blotting and in situ hybridization have revealed a high level of LRP5 expression in hepatocytes and the adrenal gland cortex. In LDLR-deficient Watanabe heritable hyperlipidemic rabbits, LRP5 mRNA is increased in the liver and accumulates in cholesterol-laden foam cells of atherosclerotic lesions (Kim, 1998).
Molecular cloning and initial functional characterization of a novel member of the low density lipoprotein receptor (LDLR) gene family is reported. The cDNA was isolated from a human osteoblast cDNA library and encodes a 1,615 amino acids protein designated as LR3. It has, in the extracellular region, a cluster of three LDLR ligand binding repeats at a juxtamembrane position and four EGF precursor homology domains separated by YWTD spacer repeats. The entire ectodomain shares the same modular organization with the middle portion of the extracellular regions of two LDLR family members, LDLR-related protein (LRP), and gp330/megalin. LR3 mRNA is expressed in most of the adult and fetal tissues examined. The highest expression level is seen in aorta. In human osteosarcoma cells examined, LR3 mRNA is highly enriched in TE85 cells, moderately expressed in MG63 cells and primary human osteoblasts, and undetectable in SaOS-2 cells. NIH 3T3 cells transfected with either full length LR3 or its ectodomain show significantly increased proliferation, whereas transfection of intracellular domain has no proliferative effect. It is predicted that LR3 is a multi-functional protein with potential mitogenic activity (Dong, 1998).
A novel secreted molecule, Wise, has been isolated by a functional screen for activities that alter the anteroposterior character of neuralized Xenopus animal caps. Wise encodes a secreted protein capable of inducing posterior neural markers at a distance. Phenotypes arising from ectopic expression or depletion of Wise resemble those obtained when Wnt signalling is altered. In animal cap assays, posterior neural markers can be induced by Wnt family members, and induction of these markers by Wise requires components of the canonical Wnt pathway. This indicates that in this context Wise activates the Wnt signalling cascade by mimicking some of the effects of Wnt ligands. Activation of the pathway was further confirmed by nuclear accumulation of ß-catenin driven by Wise. By contrast, in an assay for secondary axis induction, extracellularly Wise antagonizes the axis-inducing ability of Wnt8. Thus, Wise can activate or inhibit Wnt signalling in a context-dependent manner. The Wise protein physically interacts with the Wnt co-receptor, lipoprotein receptor-related protein 6 (LRP6), and is able to compete with Wnt8 for binding to LRP6. These activities of Wise provide a new mechanism for integrating inputs through the Wnt coreceptor complex to modulate the balance of Wnt signalling (Itasaki, 2003).
The predicted Wise protein consists of 206 amino acids and contains a cysteine knot-like domain found in a number of growth factors, as well as in Slit, mucin and CCN (Cef10/Cyr61, CTGF and Nov) family members. Among these, the C-terminal domain of the CCN family members shows the highest homology to Wise, but other motifs conserved within the CCN family are absent in Wise. Hence, Wise is related to but not a member of the CCN family. A homology search revealed that Wise shows the highest amino acid identity (38%) to Sclerostin (SOST), identified by positional cloning of the gene mutated in sclerosteosis. There are a number of EST sequences homologous to Wise in zebrafish, mouse and human databases, but none was found in the Drosophila or C. elegans genomes (Itasaki, 2003).
The Frizzled (Fz) family of serpentine receptors function as Wnt receptors, but how Fz proteins transduce signaling is not understood. Drosophila arrow encodes a transmembrane protein that is homologous to two members of the mammalian low-density lipoprotein receptor (LDLR)-related protein (LRP) family, LRP5 and LRP6. LRP6 functions as a co-receptor for Wnt signal transduction. In Xenopus embryos, LRP6 activates Wnt-Fz signaling, and induces Wnt responsive genes, dorsal axis duplication and neural crest formation. An LRP6 mutant lacking the carboxyl intracellular domain blocks signaling by Wnt or Wnt-Fz, but not by Dishevelled or beta-catenin, and inhibits neural crest development. The extracellular domain of LRP6 binds Wnt-1 and associates with Fz in a Wnt-dependent manner. These results indicate that LRP6 may be a component of the Wnt receptor complex (Tamai, 2000).
Human LRP5 and LRP6 share 71% amino-acid identity, and together with Arrow, form a distinct subgroup of the LRP family. Arrow, LRP5 and LRP6 each contain an extracellular domain with EGF (epidermal growth factor) repeats and LDLR repeats, followed by a transmembrane region and a cytoplasmic domain lacking recognizable catalytic motifs. An lrp6 mutation in mice results in pleiotropic defects recapitulating some, but not all, of the wnt mutant phenotype (Pinson, 2000). To study LRP5/LRP6 involvement in Wnt signaling, their function was examined in Wnt-induced axis and neural crest formation in Xenopus embryos (Tamai, 2000).
Wnt/beta-catenin signaling induces dorsal axis formation through activation of responsive genes, including nodal-related 3 (Xnr3) and siamois (sia). Ventral injection of LRP6 RNA into four-cell stage embryos results in dorsal axis duplication in a dose-dependent manner. In animal pole explants, LRP6 induces Xnr3/sia, but not brachyury (Xbra) expression, which is activated by mesoderm inducers like activin or basic fibroblast growth factor (bFGF). These results indicate that overexpression of LRP6 may specifically activate Wnt signaling. To examine whether LRP6 mediates Wnt effect, RNAs for LRP6 and Wnt-5a were co-injected. Neither Wnt-5a nor a low dose of LRP6 alone exhibits any effect, but Wnt-5a plus LRP6 synergistically induce axis duplication and ectopic Xnr3 expression in the embryo, and activate Xnr3/sia in explants. Synergy is also observed between Wnt-5a and hFz5, and between LRP6 and hFz5. Although LRP5 alone does not induce axes, co-injecting LRP5 and Wnt-5a does. LDLR alone or in combination with Wnt-5a does not induce axes or Xnr3/sia. Although Wnt-5a-hFz5 can induce complete axes including head and the notochord, Wnt-5a-LRP6 or LRP6 alone (higher doses) induce trunk axis with muscle and neural tissues but lacking head and the notochord. This may be explained by quantitative or qualitative differences between Wnt-5a/LRP6 and Wnt-5a/hFz5 co-injections (Tamai, 2000).
Because ectopic Wnt expression enhances, whereas lack of Wnt signaling inhibits, neural crest formation, the effect of LRP6 on neural crest development was analyzed. LDLR injection has no effect, but LRP6 expression significantly expands neural crest progenitors in the injected half of the embryo, as determined by the expression of a crest-specific marker, slug. Thus, overexpression of LRP6 also mimics Wnt signaling during neural crest formation (Tamai, 2000).
To distinguish whether LRP6 functions in Wnt-responding or Wnt-producing cells, Wnt-5a and LRP6 were injected separately into neighboring blastomeres at the four-cell stage. Induction of secondary axes in embryos and of Xnr3/sia in explants occurs even when Wnt-5a and LRP6 are expressed in different cells. Therefore, LRP6 is probably involved in responding to, rather than in enhancing, the production or secretion of the Wnt ligand (Tamai, 2000).
LRP6deltaC, which has most of its cytoplasmic domain deleted, was generated to inhibit the function of endogenous LRP6, which is expressed maternally and throughout embryogenesis. LRP6deltaC does not, either alone or in combination with Wnt-5a, induce axes or activate Xnr3/sia, but inhibits axis duplication and Xnr3/sia induction by the wild-type LRP6. This inhibition is counteracted by an increasing amount of co-injected LRP6. These data suggest that LRP6deltaC is a dominant interfering mutant for LRP6 or related molecules, and that LRP6 cytoplasmic domain is required for Wnt signaling. LRP6deltaC inhibits Xnr3/sia induction by several Wnt molecules, including Wnt-1, Wnt-2, Wnt-3a and Wnt-8. LRP6deltaC also inhibits Wnt-5a signaling through hFz5, showing that hFz5, and probably other endogenous Fz molecules mediating Wnt-1 or Wnt-8 signaling, depend on LRP6 or related proteins. LRP6deltaC does not affect Xbra induction by activin or bFGF, and thus interferes specifically with Wnt signaling (Tamai, 2000).
LRP6deltaC injected dorsally at the four-cell stage does not perturb endogenous axis formation. This may mean that the dorsal beta-catenin pathway is activated by mechanisms other than Wnt stimulation; alternatively, the dorsal Wnt-Fz signaling may occur early before LRP6deltaC can interfere. However, LRP6deltaC inhibits neural crest development as examined by slug expression, and suppresses ectopic crest formation induced by Wnt-3a DNA. Furthermore, co-injection of LRP6 rescues LRP6deltaC inhibition of crest formation. Thus, LRP6 or a related molecule is required for Wnt-dependent neural crest formation in vivo (Tamai, 2000).
In the current model of Wnt/beta-catenin signaling, Wnt stimulation of a Fz receptor activates the intracellular protein Dishevelled (Dsh or Dvl), thereby antagonizing the inhibitory action of the Axin/GSK-3 complex and stabilizing beta-catenin, which together with the transcription factor TCF/LEF activates responsive genes. To position LRP6 in this cascade, relationships between LRP6 and other Wnt signaling components were tested. LRP6deltaC inhibits Xnr3/sia induction by Wnts, but not by Dsh or beta-catenin, suggesting that LRP6deltaC interfers with Wnt signaling upstream of Dsh function. Supporting this epistasis, LRP6 induction of Xnr3/sia is antagonized by Axin, by a dominant-negative TCF, deltaNTCF, and by a dominant-negative Dsh, mDvl2-DIX. LRP6 activity is also inhibited by frizzled-related protein (FRP), a secreted Wnt antagonist, implying that LRP6 activation of Wnt signaling relies on endogenous Wnt molecules. Alternatively, FRP may directly inhibit LRP6. Thus, LRP6 acts between the extracellular Wnt, FRP and intracellular Dsh, possibly as a co-receptor for Wnt molecules (Tamai, 2000).
To function as a Wnt co-receptor, LRP6 should bind Wnt or Fz or both. Used to examine the issue were a secreted form of mFz8, mFz8CRD-IgG, comprising the cysteine-rich domain (CRD) of mFz8 N-terminal extracellular region fused with the immunoglobulin-gamma (IgG) Fc epitope, and a secreted LRP6N-Myc, consisting of the LRP6 extracellular domain tagged by the Myc epitope. mFz8CRD-IgG and LRP6N-Myc proteins were incubated with or without Wnt-1. mFz8CRD-IgG co-precipitates LRP6N-Myc only in the presence of Wnt-1; the secreted IgG fusion partner fails to do so regardless of Wnt-1. A reciprocal precipitation was also performed using secreted LRP6N-IgG and mFz8CRD-Myc, which were generated by swapping the two epitopes. LRP6N-IgG co-precipitates mFz8CRD-Myc, again only in the presence of Wnt-1, whereas the control IgG does not. LRP6N-IgG also co-precipitates Wnt-1-Myc, a tagged Wnt-1 protein. These results suggest that the extracellular domain of LRP6 can bind Wnt-1 and form a complex with Fz in a Wnt-dependent fashion (Tamai, 2000).
Thus, in two developmental processes dependent on the Wnt pathway in Xenopus -- secondary axis and neural crest formation -- LRP6 activates Wnt signaling, but a dominant-negative LRP6 inhibits Wnt signaling, providing compelling evidence that LRP6 is critical in Wnt signal transduction. LRP6 functions upstream of Dsh in Wnt-responding cells, synergizes with either Wnt or Fz, and importantly, is able to bind Wnt-1 and to associate with Fz in a Wnt-dependent manner. The simplest interpretation of these findings is that LRP6 is a component of the Wnt-Fz receptor complex. Genetic studies of arrow in Drosophila and lrp6 in mice (Pinson, 2000) strongly support this hypothesis. The binding data also raise the possibility that Wnt-induced formation of the Fz-LRP6 complex assembles LRP6, Fz and their associated proteins, thereby initiating cytoplasmic signaling. Consistent with this notion, Wnt signal transduction requires intracellular regions of both Fz and LRP6, which harbours candidate protein-protein interaction motifs. Notably, arrow does not exhibit fz planar polarity phenotype, implying that Arrow-LRP6 may specify Wnt-Fz signaling towards the beta-catenin pathway. How Fz, LRP6 and proteoglycan molecules such as Dally interact to mediate Wnt recognition/specificity and signal transduction remains to be studied. In addition, whether other LRPs and LRP-binding proteins participate in or modulate different Wnt-Fz signaling pathways needs evaluation (Tamai, 2000).
To understand how the Wnt coreceptor LRP-5 is involved in transducing the canonical Wnt signals, Axin was identified as a protein that interacts with the intracellular domain of LRP-5. LRP-5, when expressed in fibroblast cells, shows no effect on the canonical Wnt signaling pathway by itself, but instead acts synergistically with Wnt. In contrast, LRP-5 mutants that lack the extracellular domain function as constitutively active forms that bind Axin and that induce LEF-1 activation by destabilizing Axin and stabilizing beta-catenin. Addition of Wnt causes the translocation of Axin to the membrane and enhances the interaction between Axin and LRP-5. In addition, the LRP-5 sequences involved in interactions with Axin are required for LEF-1 activation. Thus, it is concluded that the binding of Axin to LRP-5 is an important part of the Wnt signal transduction pathway (J. Mao, 2001).
Thus, expression of the cytoplasmic domain of LRP-5, with or without the transmembrane domain, acts as a constitutive activator of the Wnt pathway, activating LEF-1 transcription and stabilizing ß-catenin. This result suggests that the extracellular domain inhibits the function of the intracellular domain. Binding of a Wnt ligand presumably overcomes this inhibition, perhaps by inducing a conformational change in LRP-5 or through interactions with the Frizzled receptor. Intriguingly, a construct containing the intracellular domain and the transmembrane region (LRPC2) was significantly more active than one containing just the intracellular domain (LRPC3), indicating that recruitment of Axin to the membrane is important for the activation of the Wnt pathway by LRP-5. The finding that a LRPC3 variant containing a myristylation signal shows an activity similar to LRPC2 lends further supports to this idea. One of the roles that the translocation plays is Axin destabilization, which has previously been shown to be induced by Wnt, because expression of LRPC2, but not LRPC3, caused Axin degradation. Together with the results showing that Wnt stimulation causes Axin to be recruited to the membrane via LRP-5, it is proposed that Wnt induces Axin destabilization at least in part by stimulating the interaction of LRP-5 with Axin. Although the precise mechanism by which activated LRP-5 causes the destabilization of Axin is not clear, it is reasonable to conclude that Axin destabilization contributes to the stabilization of ß-catenin (J. Mao, 2001).
The degradation of Axin is not the only means of inhibiting its function. This is consistent with the results that a construct that does not degrade Axin (LRPC3) can still stimulate LEF-1 transcription. Thus, part of the mechanism by which the intracellular domain of LRP-5 inhibits the function of Axin may simply involve the binding of this region to Axin, thus preventing it from participating in the degradation of ß-catenin. The region necessary for binding Axin and for stimulating LEF-1 transcription was narrowed down to 40 C-terminal amino acids in LRP-5. This region contains three copies of a motif PPT/SP, which is conserved in human and mouse LRP-5 and LRP-6 and Drosophila Arrow. Although removal of the very C-terminal motif shows little effect, elimination of two of these motifs reduces both Axin binding and LEF-1 stimulation but still retains some activity. Truncation of an additional 10 amino acids, including the third motif, abolishes Axin binding and transcriptional activation. This repeated motif could function as a phosphorylation site for a serine/threonine kinase. In this light, it is interesting that the wild-type GSK3 strongly stimulates the binding of Axin to LRP-5, whereas a kinase-dead GSK3 does not. While the intracellular domain of LRP-5 cannot be phosphorylated by GSK3 when immunoprecipitated LRPC2 and GSK were tested in an in vitro kinase assay, GSK3 may stimulate an additional kinase. Alternatively, the phosphorylation of Axin by GSK3 might cause it to bind LRP-5 more effectively. For instance, the phosphorylation of Axin by GSK3 enhances Axin's ability to bind ß-catenin and to bind GSK3 itself. Given that GSK3 promotes ß-catenin degradation, while the LRP/Axin interaction has the opposite result, the stimulatory effect of GSK3 on the interaction between Axin and LRP-5 is somewhat surprising. However, such an effect may allow LRP-5 to interact only with the Axin molecules that are associated with GSK3. In this way, LRP-5 could specifically direct the degradation of only those (GSK3-associated) Axin molecules that would otherwise participate in the degradation of ß-catenin (J. Mao, 2001).
Wnt-3a-induced binding of Axin to LRP-5 occurs within 4 min of the addition of ligand. This ligand-induced process occurs much sooner than the other processes that have thus far been reported. Wnt proteins induce the dephosphorylation of Axin and Dvl within 30 min, and the degradation of Axin starting at approximately 2 hr. Thus, the rapid stimulation of the interaction of LRP-5 and Axin by Wnt-3a suggests that the interaction between LRP-5 and Axin might be one of the first events in the Wnt canonical pathway (J. Mao, 2001).
In summary, a model is proposed describing the involvement of LRP proteins in the canonical Wnt signaling pathway. In this model, it is suggested that LRP, when activated by Wnt proteins, recruits Axin to the membranes. The translocation of Axin to the membrane prevents it from participating in the degradation of ß-catenin and plays an important role in the destabilization of Axin. LRP-6, a close homolog of LRP-5, may act in the same way as LRP-5, because a LRP-6 mutant that is equivalent to LRPC2 can also bind Axin and constitutively activates LEF-1. Thus, binding to Axin and activation of the canonical Wnt signaling pathway may occur with other LRP-5 homologs, including Arrow (J. Mao, 2001).
Although many components of the Wnt signaling pathway have been identified, little is known about how Wnts and their cognate Frizzled receptors signal to downstream effector molecules. Evidence is presented that a new member of the low-density lipoprotein (LDL)-receptor-related protein family, LRP6, is critical for Wnt signaling in mice. Embryos homozygous for an insertion mutation in the LRP6 gene exhibit developmental defects that are a striking composite of those caused by mutations in individual Wnt genes. Furthermore, a genetic enhancement of a Wnt mutant phenotype has been shown in mice lacking one functional copy of LRP6. These results support a broad role for LRP6 in the transduction of several Wnt signals in mammals (Pinson, 2000).
In a screen for recessive lethal phenotypes in mice caused by gene trap insertions in cell-surface proteins, an insertion mutation was recovered that joined the first 321 amino acids of the LRP6 protein in-frame with the betageo reporter gene. Embryos homozygous for the insertion in LRP6 die at birth, and exhibit a variety of severe developmental abnormalities including a truncation of the axial skeleton, limb defects, microophthalmia and malformation of the urogenital system. Northern blot analysis shows a complete absence of LRP6 transcripts in LRP6-/- embryos and embryonic fibroblasts. Southern blot analysis indicates a simple insertion event in which LRP6 exons downstream of the vector are retained. On the basis of betageo reporter activity, which accurately reports endogenous gene expression, the LRP6 gene appears to be expressed in all cells of the developing embryo (Pinson, 2000).
The specific developmental defects that are observed in homozygous embryos are remarkably similar to mice carrying mutations in Wnt genes, specifically Wnt-3a, Wnt-1 and Wnt-7a. Both the targeted null mutation and a classical hypomorphic allele of Wnt-3a, vestigial tail (vt), cause caudal truncations of the body axis. A similar axial truncation was observed in LRP6-/- neonates in which vertebrae caudal to the lumbar regions are absent. This was first evident as a reduction in the size of the tailbud at 8.5 days of development, with the loss of paraxial mesoderm and caudal somites at later stages. Sections through the tailbud of LRP6-/- embryos revealed an excess of neural tissue and a corresponding loss of paraxial mesoderm, similar to what has been observed in the targeted Wnt-3a mutant. As in vt mutants, Wnt-3a transcripts are reduced in the tailbud. By 10.5 days of development, Wnt-3a transcripts are completely absent from the tail, probably reflecting the depletion of mesodermal precursors at this stage. The extent of axial truncations in vt and Wnt-3a null mutants is sensitive to the dose of Wnt-3a. On the basis of the level at which the axial skeleton is truncated, it is concluded that LRP6-/- embryos display a severely hypomorphic Wnt-3a phenotype (Pinson, 2000).
To determine whether the vestigial tail phenotype is modified by the dose of LRP6, vt;LRP6 double heterozygous animals were crossed to vt/vt animals and embryos were collected at 11.5 and 13.5 days of development. The expected ratio of phenotypic classes was observed: half showed a normal tail length (corresponding to the vt/+;+/+ and vt/+;LRP6-/+ genotypes) and half exhibited a truncation of the tail (representing the vt/ vt;+/+ and vt/vt;LRP6-/+ genotypes). The tail defects in vt/vt embryos were clearly more pronounced among all eight embryos that inherited the LRP6 mutant allele. The enhancement of the vestigial tail phenotype upon elimination of one functional copy of the LRP6 gene provides genetic evidence that LRP6 and Wnt-3a function in the same pathway (Pinson, 2000).
Mutant embryos were examined for mid/hindbrain defects, a hallmark of Wnt-1-deficient mice. The phenotypes of a classical Wnt-1 allele, swaying, and a targeted mutation of Wnt-1 have variable expressivity, exhibiting a range of malformations of the midbrain and cerebellum. About one-half of the LRP6 mutants exhibit neural tube closure defects (exencephaly and/or spina bifida), and among those embryos that were not exencephalic morphological changes at the mid/hindbrain junction were observed. At 10.5 days, the mid/hindbrain boundary is less distinct in LRP6 mutant embryos, and by 14.5 days, a deletion of the caudal midbrain is evident. At birth, the inferior colliculus is absent and the cerebellum is disorganized. Furthermore, an elevation and expansion of Wnt-1 transcripts observed in swaying embryos is also seen in LRP6-/- embryos. Thus, LRP6-deficient mice phenocopy the less severe class of mid/hindbrain defects observed in Wnt-1 mutant mice (Pinson, 2000).
LRP6 mutant embryos display both anteroposterior and dorsoventral patterning defects in the limbs. These defects are reminiscent of those observed in Wnt-7a mutant mice that fail to maintain Shh expression in the zone of polarizing activity (ZPA), leading to the variable loss of posterior digits. Wnt-7a is also required to establish dorsal cell fates in the limb: in its absence, a biventral limb is formed. Because hindlimb defects in the LRP6 mutants may be exacerbated by a general disruption of caudal development, an analysis was focused on forelimb development. The forelimbs of LRP6 -/- embryos show a loss of one or more posterior digits that is presaged at earlier stages by a gradual loss of Shh expression. Moreover, some of the remaining digits contained ectopic tendons diagnostic of a ventralized limb. A distinguishing feature of the LRP6 limb phenotype not observed in Wnt-7a mutants was a failure to maintain the apical ectodermal ridge (AER), as judged by discontinuous expression of Fgf8. Discontinuity of the AER may explain the variable loss of anterior digits observed in LRP6 mutant embryos. The added complexity may indicate that, in addition to Wnt-7a, LRP6 mediates signaling by other Wnts in the limb (Pinson, 2000).
Clearly, LRP6 mutant mice do not exhibit all of the Wnt phenotypes reported to date (for example, the early embryonic lethality associated with Wnt-3 mutants and the limb outgrowth defects in Wnt-5a mutants; therefore, LRP6 may be involved in signaling by only a subset of vertebrate Wnts. Furthermore, the defects in LRP6 mutant embryos are generally less severe than those exhibited by individual Wnt mutants. Notably, a second highly related family member, LRP5, is widely expressed in embryos and may limit the range and severity of Wnt phenotypes observed in LRP6-/- mice (Pinson, 2000).
Members of the LDL receptor family are classically known for their involvement in the endocytic pathway, mediating the uptake through clathrin-coated pits of macromolecules such as apolipoproteins, lipases and proteases. Mice carrying mutations in two members of the LDL receptor family (the VLDL and ApoER2 receptors) have been shown to phenocopy mouse mutations in reelin or disabled , providing the first indication that LDL receptors can participate in specific signaling pathways. Given the central importance of the LDL receptor family in the transport of lipoproteins and other macromolecules into the cell, the relationship between nutrient uptake and the response of cells to developmental signals such as Wnts warrants further investigation (Pinson, 2000).
Wnt glycoproteins have been implicated in diverse processes during embryonic patterning in metazoa. They signal through frizzled-type seven-transmembrane-domain receptors to stabilize beta-catenin. Wnt signalling is antagonized by the extracellular Wnt inhibitor dickkopf1 (dkk1), which is a member of a multigene family. dkk1 was initially identified as a head inducer in Xenopus embryos but the mechanism by which it blocks Wnt signalling is unknown. LDL-receptor-related protein 6 (LRP6) is required during Wnt/beta-catenin signalling in Drosophila, Xenopus and mouse, possibly acting as a co-receptor for Wnt. LRP6 is a specific, high-affinity receptor for Dkk1 and Dkk2. Dkk1 blocks LRP6-mediated Wnt/beta-catenin signalling by interacting with domains that are distinct from those required for Wnt/Frizzled interaction. dkk1 and LRP6 interact antagonistically during embryonic head induction in Xenopus where LRP6 promotes the posteriorizing role of Wnt/beta-catenin signalling. Thus, DKKs inhibit Wnt co-receptor function, exemplifying the modulation of LRP signalling by antagonists (B. Mao, 2001).
The differentiation of epithelial cells and fiber cells from the anterior and posterior compartments of the lens vesicle, respectively, give the mammalian lens its distinctive polarity. While much progress has been made in understanding the molecular basis of fiber differentiation, little is known about factors that govern the differentiation of the epithelium. Members of the Wnt growth factor family appear to be key regulators of epithelial differentiation in various organ systems. Wnts are ligands for Frizzled receptors and can activate several signaling pathways, of which the best understood is the Wnt/ß-catenin pathway. The presence of LDL-related protein coreceptors (LRPs) 5 or 6 has been shown to be a requirement for Wnt signaling through the ß-catenin pathway. To access the role of this signaling pathway in the lens, mice were analyzed with a null mutation of lrp6. These mice have small eyes and aberrant lenses, characterized by an incompletely formed anterior epithelium resulting in extrusion of the lens fibers into the overlying corneal stroma. Multiple Wnts, including 5a, 5b, 7a, 7b, 8a, 8b, and Frizzled receptors 1, 2, 3, 4, and 6, are detected in the lens. Expression of these molecules is generally present throughout the lens epithelium and extended into the transitional zone, where early fiber elongation occurs. In addition to both LRP5 and LRP6, the expression was shown of other molecules involved in Wnt signaling and its regulation, including Dishevelleds, Dickkopfs, and secreted Frizzled-related proteins. Taken together, these results indicate a role for Wnt signaling in regulating the differentiation and behavior of lens cells (Stump, 2003).
Current models of canonical Wnt signaling assume that a pathway is active if β-catenin becomes nuclearly localized and Wnt target genes are transcribed. In Xenopus, maternal LRP6 is essential in such a pathway, playing a pivotal role in causing expression of the organizer genes siamois and Xnr3, and in establishing the dorsal axis. Evidence iis provided that LRP6 acts by degrading axin protein during the early cleavage stage of development. In the full-grown oocyte, before maturation, axin levels are also regulated by Wnt11 and LRP6. In the oocyte, Wnt11 and/or LRP6 regulates axin to maintain β-catenin at a low level, while in the embryo, asymmetrical Wnt11/LRP6 signaling stabilizes β-catenin and enriches it on the dorsal side. This suggests that canonical Wnt signaling may not exist in simple off or on states, but may also include a third, steady-state, modality (Kofron, 2007)
Recent work has identified LDL receptor-related family members, Lrp5 and Lrp6, as co-receptors for the transduction of Wnt signals. Analysis of mice carrying mutations in both Lrp5 and Lrp6 demonstrates that the functions of these genes are redundant and are essential for gastrulation. Lrp5;Lrp6 double homozygous mutants fail to establish a primitive streak, although the anterior visceral endoderm and anterior epiblast fates are specified. Thus, Lrp5 and Lrp6 are required for posterior patterning of the epiblast, consistent with a role in transducing Wnt signals in the early embryo. Interestingly, Lrp5+/-;Lrp6-/- embryos die shortly after gastrulation and exhibit an accumulation of cells at the primitive streak and a selective loss of paraxial mesoderm. A similar phenotype is observed in Fgf8 and Fgfr1 mutant embryos and provides genetic evidence in support of a molecular link between the Fgf and Wnt signaling pathways in patterning nascent mesoderm. Lrp5+/-;Lrp6-/- embryos also display an expansion of anterior primitive streak derivatives and anterior neurectoderm that correlates with increased Nodal expression in these embryos. The effect of reducing, but not eliminating, Wnt signaling in Lrp5+/-;Lrp6-/- mutant embryos provides important insight into the interplay between Wnt, Fgf and Nodal signals in patterning the early mouse embryo (Kelly, 2004).
Wnt signaling regulates β-catenin-mediated gene transcription and planar cell polarity (PCP). The Wnt co-receptor, Lrp6, is required for signaling along the β-catenin arm. Lrp6 downregulation (by morpholino injection) or overexpression in Xenopus embryos disrupts convergent extension, a hallmark feature of Wnt/PCP components. In embryos with decreased Lrp6 levels, cells of the dorsal marginal zone (DMZ), which undergoes extensive cellular rearrangements during gastrulation, exhibit decreased length:width ratios, decreased migration, and increased numbers of transient cytoplasmic protrusions. Lrp6 opposes Wnt11 activity and localizes to the posterior edge of migrating DMZ cells; Lrp6 downregulation enhances cortical and nuclear localization of Dsh and phospho-JNK, respectively. Taken together, these data suggest that Lrp6 inhibits Wnt/PCP signaling. Finally, the region of the Lrp6 protein with Wnt/PCP activity was localized to a stretch of 36 amino acids, distinct from regions required for Wnt/β-catenin signaling. A model is proposed in which Lrp6 plays a critical role in the switch from Wnt/PCP to Wnt/β-catenin signaling (Tahinci, 2007)
Dickkopf-1 (Dkk1) is a secreted protein that negatively modulates the Wnt/βcatenin pathway. Lack of Dkk1 function affects head formation in frog and mice, supporting the idea that Dkk1 acts as a 'head inducer' during gastrulation. Lack of Dkk1 function accelerates internalization and rostral progression of the mesendoderm and gain of function slows down both internalization and convergence extension, indicating a novel role for Dkk1 in modulating these movements. The motility phenotype found in the morphants is not observed in embryos in which the Wnt/βcatenin pathway is overactivated, and dominant-negative Wnt proteins are not able to rescue the gastrulation movement defect induced by absence of Dkk1. These data strongly suggest that Dkk1 is acting in a βcatenin independent fashion when modulating gastrulation movements. The glypican 4/6 homolog Knypek (Kny) binds to Dkk1, and they are able to functionally interact in vivo. Moreover, Dkk1 regulation of gastrulation movements is kny dependent. Kny is a component of the Wnt/planar cell polarity (PCP) pathway. Indeed Dkk1 is able to activate this pathway in both Xenopus and zebrafish. Furthermore, concomitant alteration of the βcatenin and PCP activities is able to mimic the morphant accelerated cell motility phenotype. These data therefore indicate that Dkk1 regulates gastrulation movement through interaction the Frizzled coreceptor transmembrane protein LRP5/6 and Kny and coordinated modulations of Wnt/βcatenin and Wnt/PCP pathways (Caneparo, 2007).
Specification of embryonic polarity and pattern formation in multicellular organisms requires inductive signals from neighboring cells. One approach toward understanding these interactions is to study mutations that disrupt development. mesd, a gene identified in the mesoderm development (mesd) deletion interval on mouse chromosome 7, is essential for specification of embryonic polarity and mesoderm induction. MESD functions in the endoplasmic reticulum as a specific chaperone for LRP5 and LRP6, which in conjunction with Frizzled, are coreceptors for canonical WNT signal transduction. Disruption of embryonic polarity and mesoderm differentiation in mesd-deficient embryos likely results from a primary defect in WNT signaling. However, phenotypic differences between mesd-deficient and wnt3 minus embryos suggest that MESD may function on related members of the low-density lipoprotein receptor (LDLR) family, whose members mediate diverse cellular processes ranging from cargo transport to signaling (Hsieh, 2003).
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