Wg Protein Research Articles

Leucine improves dietary protein use efficiency by regulating protein synthesis by activating amino acid transporters and the mTORC1 pathways in Chinese mitten crab (Eriocheir sinensis)

Leucine plays a critical role in protein metabolism and energy homeostasis. This study determines whether leucine supplementation increases body protein synthesis in juvenile E. sinensis while consuming less protein than their requirements. It also investigates the effects of leucine on molecular mechanisms regulating protein synthesis. The 8-week growth trial included three dietary protein levels (22%, 30% or 38%) crossing three leucine levels (1.1, 2.5 or 5.0%). In the trial, 1440 crabs (0.40 ± 0.05 g) were allocated to a 3 × 3 factorial experiment with four replicates of 40 crabs each. The WG and SGR of the NP group were significantly higher than those of the LLP group when the diet contained 1.1% leucine (P < 0.05). The WG, SGR and whole-body protein content of the LP group were significantly higher than those of the LLP group with 2.5% leucine added to the diet (P < 0.05) but not significantly different from those of the NP group (P > 0.05). However, when 5% leucine was added to the diet, the whole-body protein content of the LP group was significantly higher than that of the LLP group (P < 0.05). Dietary leucine supplementation significantly improved growth performance, increased crude protein and decreased crude lipids in the crabs. When the diet contained 30% crude protein, the highest WG, SGR, PER and PRR appeared in the LP-2.5 group (P > 0.05). The PER and PRR of the NP-2.5 group were significantly higher than those of the NP-1.1 group (P < 0.05) and were not significantly different from those of the NP-5.0 group (P > 0.05). In addition, the whole-body protein content of the NP-2.5 group was significantly higher than that of the NP-1.1 and NP-5.0 groups (P < 0.05). Growth hormone and ecdysterone significantly increased with dietary protein levels. The highest growth hormone and ecdysterone in the hemolymph occurred in the 2.5% leucine groups. Moreover, leucine supplementation significantly increased lipase and amylase activity in the hepatopancreas (P < 0.05). The 2.5% leucine supplementation significantly increased CAT activity and decreased MDA in the hepatopancreas. Dietary leucine significantly increased hemocyanin in the hemolymph, and the expression of amino acid transporter genes was significantly increased with dietary leucine supplementation. The crabs fed 2.5% leucine supplementation had the highest gene expression of the mTOR pathway, indicating that dietary leucine significantly promotes amino acid transport and protein synthesis in the hepatopancreas and muscles. This study suggests that a low-protein diet can inhibit animal protein synthesis and decrease energy accumulation and growth. The 2.5% leucine supplementation in the 30% protein diet effectively could promote protein synthesis and utilization and improve crabs growth and protein utlization.

Dynein Heavy Chain 64C Differentially Regulates Cell Survival and Proliferation of Wingless-Producing Cells in Drosophila melanogaster.

Dynein is a multi-subunit motor protein that moves toward the minus-end of microtubules, and plays important roles in fly development. We identified Dhc64Cm115, a new mutant allele of the fly Dynein heavy chain 64C (Dhc64C) gene whose heterozygotes survive against lethality induced by overexpression of Sol narae (Sona). Sona is a secreted metalloprotease that positively regulates Wingless (Wg) signaling, and promotes cell survival and proliferation. Knockdown of Dhc64C in fly wings induced extensive cell death accompanied by widespread and disorganized expression of Wg. The disrupted pattern of the Wg protein was due to cell death of the Wg-producing cells at the DV midline and overproliferation of the Wg-producing cells at the hinge in disorganized ways. Coexpression of Dhc64C RNAi and p35 resulted in no cell death and normal pattern of Wg, demonstrating that cell death is responsible for all phenotypes induced by Dhc64C RNAi expression. The effect of Dhc64C on Wg-producing cells was unique among components of Dynein and other microtubule motors. We propose that Dhc64C differentially regulates survival of Wg-producing cells, which is essential for maintaining normal expression pattern of Wg for wing development.

Extracellular spreading of Wingless is required forDrosophilaoogenesis.

Recent studies have investigated whether the Wnt family of extracellular ligands can signal at long range, spreading from their source and acting as morphogens, or whether they signal only in a juxtacrine manner to neighboring cells. The original evidence for long-range Wnt signaling arose from studies of Wg, a Drosophila Wnt protein, which patterns the wing disc over several cell diameters from a central source of Wg ligand. However, the requirement of long-range Wg for patterning was called into question when it was reported that replacing the secreted protein Wg with a membrane-tethered version, NRT-Wg, results in flies with normally patterned wings. We and others previously reported that Wg spreads in the ovary about 50 μm or 5 cell diameters, from the cap cells to the follicle stem cells (FSCs) and that Wg stimulates FSC proliferation. We used the NRT-wg flies to analyze the consequence of tethering Wg to the cap cells. NRT-wg homozygous flies are sickly, but we found that hemizygous NRT-wg/null flies, carrying only one copy of tethered Wingless, were significantly healthier. Despite their overall improved health, these hemizygous flies displayed dramatic reductions in fertility and in FSC proliferation. Further, FSC proliferation was nearly undetectable when the wg locus was converted to NRT-wg only in adults, and the resulting germarium phenotype was consistent with a previously reported wg loss-of-function phenotype. We conclude that Wg protein spreads from its source cells in the germarium to promote FSC proliferation.

The Impact of Drosophila Awd/NME1/2 Levels on Notch and Wg Signaling Pathways.

Awd, the Drosophila homologue of NME1/2 metastasis suppressors, plays key roles in many signaling pathways. Mosaic analysis of the null awdJ2A4 allele showed that loss of awd gene function blocks Notch signaling and the expression of its target genes including the Wingless (Wg/Wnt1) morphogen. We also showed that RNA interference (RNAi)-mediated awd silencing (awdi) in larval wing disc leads to chromosomal instability (CIN) and to Jun amino-terminal kinases (JNK)-mediated cell death. Here we show that this cell death is independent of p53 activity. Based on our previous finding showing that forced survival of awdi-CIN cells leads to aneuploidy without the hyperproliferative effect, we investigated the Wg expression in awdi wing disc cells. Interestingly, the Wg protein is expressed in its correct dorso-ventral domain but shows an altered cellular distribution which impairs its signaling. Further, we show that RNAi-mediated knock down of awd in wing discs does not affect Notch signaling. Thus, our analysis of the hypomorphic phenotype arising from awd downregulation uncovers a dose-dependent effect of Awd in Notch and Wg signaling.

Wingless and Archipelago, a fly E3 ubiquitin ligase and a homolog of human tumor suppressor FBW7, show an antagonistic relationship in wing development

BackgroundArchipelago (Ago) is a Drosophila homolog of mammalian F-box and WD repeat domain-containing 7 (FBW7, also known as FBXW7). In previous studies, FBW7 has been addressed as a tumor suppressor mediating ubiquitin-dependent proteolysis of several oncogenic proteins. Ubiquitination is a type of protein modification that directs protein for degradation as well as sorting. The level of beta-catenin (β-cat), an intracellular signal transducer in Wnt signaling pathway, is reduced upon overexpression of FBW7 in human cancer cell lines. Loss of function mutations in FBW7 and overactive Wnt signaling have been reported to be responsible for human cancers.ResultsWe found that Ago is important for the formation of shafts in chemosensory bristles at wing margin. This loss of shaft phenotype by knockdown of ago was rescued by knockdown of wingless (wg) whereas wing notching phenotype by knockdown of wg was rescued by knockdown of ago, establishing an antagonistic relationship between ago and wg. In line with this finding, knockdown of ago increased the level of Armadillo (Arm), a homolog of β-cat, in Drosophila tissue. Furthermore, knockdown of ago increased the level of Distal-less (Dll) and extracellular Wg in wing discs. In S2 cells, the amount of secreted Wg was increased by knockdown of Ago but decreased by Ago overexpression. Therefore, Ago plays a previously unidentified role in the inhibition of Wg secretion. Ago-overexpressing clones in wing discs exhibited accumulation of Wg in endoplasmic reticulum (ER), suggesting that Ago prevents Wg protein from moving to Golgi from ER.ConclusionsWe concluded that Ago plays dual roles in inhibiting Wg signaling. First, Ago decreases the level of Arm, by which Wg signaling is downregulated in Wg-responding cells. Second, Ago decreases the level of extracellular Wg by inhibiting movement of Wg from ER to Golgi in Wg-producing cells.

Robust Wnt signaling is maintained by a Wg protein gradient and Fz2 receptor activity in the developing Drosophila wing.

ABSTRACTWnts are secreted proteins that regulate cell fate during development of all metazoans. Wnt proteins were proposed to spread over several cells to activate signaling directly at a distance. In the Drosophila wing epithelium, an extracellular gradient of the Wnt1 homolog Wingless (Wg) was observed extending over several cells away from producing cells. Surprisingly, however, it was also shown that a membrane-tethered Neurotactin-Wg fusion protein (NRT-Wg) can largely replace endogenous Wg, leading to proper patterning of the wing. Therefore, the functional range of Wg and whether Wg spreading is required for correct tissue patterning remains controversial. Here, by capturing secreted Wg on cells away from the source, we show that Wg acts over a distance of up to 11 cell diameters to induce signaling. Furthermore, cells located outside the reach of extracellular Wg depend on the Frizzled2 receptor to maintain signaling. Frizzled2 expression is increased in the absence of Wg secretion and is required to maintain signaling and cell survival in NRT-wg wing discs. Together, these results provide insight into the mechanisms by which robust Wnt signaling is achieved in proliferating tissues.

Regulation of the protein kinase activity of ShaggyZeste-white3 by components of the Wingless pathway in Drosophila cells and embryos.

The protein-serine kinase Shaggy(Zeste-white3) (Sgg(Zw3)) is the Drosophila homolog of mammalian glycogen synthase kinase-3 and has been genetically implicated in signal transduction pathways necessary for the establishment of patterning. Sgg(Zw3) is a putative component of the Wingless (Wg) pathway, and epistasis analyses suggest that Sgg(Zw3) function is repressed by Wg signaling. Here, we have investigated the biochemical consequences of Wg signaling with respect to the Sgg(Zw3) protein kinase in two types of Drosophila cell lines and in embryos. Our results demonstrate that Sgg(Zw3) activity is inhibited following exposure of cells to Wg protein and by expression of downstream components of Wg signaling, Drosophila frizzled 2 and dishevelled. Wg-dependent inactivation of Sgg(Zw3) is accompanied by serine phosphorylation. We also show that the level of Sgg(Zw3) activity regulates the stability of Armadillo protein and modulates the level of phosphorylation of D-Axin and Armadillo. Together, these results provide direct biochemical evidence in support of the genetic model of Wg signaling and provide a model for dissecting the molecular interactions between the signaling proteins.

A matrix metalloproteinase mediates long-distance attenuation of stem cell proliferation.

Ligand-based signaling can potentiate communication between neighboring cells and between cells separated by large distances. In the Drosophila melanogaster ovary, Wingless (Wg) promotes proliferation of follicle stem cells located ~50 µm or five cell diameters away from the Wg source. How Wg traverses this distance is unclear. We find that this long-range signaling requires Division abnormally delayed (Dally)-like (Dlp), a glypican known to extend the range of Wg ligand in the wing disc by binding Wg. Dlp-mediated spreading of Wg to follicle stem cells is opposed by the extracellular protease Mmp2, which cleaved Dlp in cell culture, triggering its relocalization such that Dlp no longer contacted Wg protein. Mmp2-deficient ovaries displayed increased Wg distribution, activity, and stem cell proliferation. Mmp2 protein is expressed in the same cells that produce Wg; thus, niche cells produce both a long-range stem cell proliferation factor and a negative regulator of its spreading. This system could allow for spatial control of Wg signaling to targets at different distances from the source.

A novel planar polarity gene pepsinogen‐like regulates wingless expression in a posttranscriptional manner

Planar cell polarity (PCP) originally referred to the coordination of global organ axes and individual cell polarity within the plane of the epithelium. More recently, it has been accepted that pertinent PCP regulators play essential roles not only in epithelial sheets, but also in various rearranging cells. We identified pepsinogen-like (pcl) as a new planar polarity gene, using Drosophila wing epidermis as a model. Pcl protein is predicted to belong to a family of aspartic proteases. When pcl mutant clones were observed in pupal wings, PCP was disturbed in both mutant and wild-type cells that were juxtaposed to the clone border. We examined levels of known PCP proteins in wing imaginal discs. The amount of the seven-pass transmembrane cadherin Flamingo (Fmi), one of the PCP "core group" members, was significantly decreased in mutant clones, whereas neither the amount of nor the polarized localization of Dachsous (Ds) at cell boundaries was affected. In addition to the PCP phenotype, the pcl mutation caused loss of wing margins. Intriguingly, this was most likely due to a dramatic decrease in the level of Wingless (Wg) protein, but not due to a decrease in the level of wg transcripts. Our results raise the possibility that Pcl regulates Wg expression post-transcriptionally, and PCP, by proteolytic cleavages.

Centrosomal kinase Nek2 cooperates with oncogenic pathways to promote metastasis

Centrosomal kinase Nek2 is overexpressed in different cancers, yet how it contributes toward tumorigenesis remains poorly understood. dNek2 overexpression in a Drosophila melanogaster model led to upregulation of Drosophila Wnt ortholog wingless (Wg), and alteration of cell migration markers—Rho1, Rac1 and E-cadherin (Ecad)—resulting in changes in cell shape and tissue morphogenesis. dNek2 overexpression cooperated with receptor tyrosine kinase and mitogen-activated protein kinase signaling to upregulate activated Akt, Diap1, Mmp1 and Wg protein to promote local invasion, distant seeding and metastasis. In tumor cell injection assays, dNek2 cooperated with Ras and Src signaling to promote aggressive colonization of tumors into different adult fly tissues. Inhibition of the PI3K pathway suppressed the cooperation of dNek2 with other growth pathways. Consistent with our fly studies, overexpression of human Nek2 in A549 lung adenocarcinoma and HEK293T cells led to activation of the Akt pathway and increase in β-catenin protein levels. Our computational approach identified a class of Nek2-inhibitory compounds and a novel drug-like pharmacophore that reversed the Nek2 overexpression phenotypes in flies and human cells. Our finding posits a novel role for Nek2 in promoting metastasis in addition to its currently defined role in promoting chromosomal instability. It provides a rationale for the selective advantage of centrosome amplification in cancer.

Sphingolipid depletion impairs endocytic traffic and inhibits Wingless signaling

Sphingolipids are an important part of the plasma membrane and implicated in a multitude of cellular processes. However, little is known about the role of sphingolipids in an epithelial context and their potential influence on the activity of signaling pathways. To shed light on these aspects we analyzed the consequences of changing ceramide levels in vivo in the Drosophila wing disc: an epithelial tissue in which the most fundamental signaling pathways, including the Wnt/Wg signaling pathway, are well characterized.We found that downregulation of Drosophila’s only ceramide synthase gene schlank led to defects in the endosomal trafficking of proteins. One of the affected proteins is the Wnt ligand Wingless (Wg) that accumulated. Unexpectedly, although Wg protein levels were raised, signaling activity of the Wg pathway was impaired. Recent work has spotlighted the central role of the endocytic trafficking in the transduction of the Wnt signal. Our results underscore this and support the view that sphingolipid levels are crucial in orchestrating epithelial endocytic trafficking in vivo. They further demonstrate that ceramide/sphingolipid levels can affect Wnt signaling.

Drosophila Heparan Sulfate 6-O-Endosulfatase Sulf1 Facilitates Wingless (Wg) Protein Degradation

Heparan sulfate proteoglycans regulate various physiological and developmental processes through interactions with a number of protein ligands. Heparan sulfate (HS)-ligand binding depends on the amount and patterns of sulfate groups on HS, which are controlled by various HS sulfotransferases in the Golgi apparatus as well as extracellular 6-O-endosulfatases called "Sulfs." Sulfs are a family of secreted molecules that specifically remove 6-O-sulfate groups within the highly sulfated regions on HS. Vertebrate Sulfs promote Wnt signaling, whereas the only Drosophila homologue of Sulfs, Sulf1, negatively regulates Wingless (Wg) signaling. To understand the molecular mechanism for the negative regulation of Wg signaling by Sulf1, we studied the effects of Sulf1 on HS-Wg interaction and Wg stability. Sulf1 overexpression strongly inhibited the binding of Wg to Dally, a potential target heparan sulfate proteoglycan of Sulf1. This effect of Drosophila Sulf1 on the HS-Wg interaction is similar to that of vertebrate Sulfs. Using in vitro, in vivo, and ex vivo systems, we show that Sulf1 reduces extracellular Wg protein levels, at least partly by facilitating Wg degradation. In addition, expression of human Sulf1 in the Drosophila wing disc lowers the levels of extracellular Wg protein, as observed for Drosophila Sulf1. Our study demonstrates that vertebrate and Drosophila Sulfs have an intrinsically similar activity and that the function of Sulfs in the fate of Wnt/Wg ligands is context-dependent.

Roles of N-glycosylation and lipidation in Wg secretion and signaling

Wnt members act as morphogens essential for embryonic patterning and adult homeostasis. Currently, it is still unclear how Wnt secretion and its gradient formation are regulated. In this study, we examined the roles of N-glycosylation and lipidation/acylation in regulating the activities of Wingless (Wg), the main Drosophila Wnt member. We show that Wg mutant devoid of all the N-glycosylations exhibits no major defects in either secretion or signaling, indicating that N-glycosylation is dispensable for Wg activities. We demonstrate that lipid modification at Serine 239 (S239) rather than that at Cysteine 93 (C93) plays a more important role in regulating Wg signaling in multiple developmental contexts. Wg S239 mutant exhibits a reduced ability to bind its receptor, Drosophila Frizzled 2 (dFz2), suggesting that S239 is involved in the formation of a Wg/receptor complex. Importantly, while single Wg C93 or Wg S239 mutants can be secreted, removal of both acyl groups at C93 and S239 renders Wg incapable of reaching the plasma membrane for secretion. These data argue that lipid modifications at C93 and S239 play major roles in Wg secretion. Further experiments demonstrate that two acyl attachment sites in the Wg protein are required for the interaction of Wg with Wntless (Wls, also known as Evi or Srt), the key cargo receptor involved in Wg secretion. Together, our data demonstrate the in vivo roles of N-glycosylation and lipid modification in Wg secretion and signaling.

Synthesis of nanocomposite films from wheat gluten matrix and MMT intercalated with different quaternary ammonium salts by way of hydroalcoholic solvent casting

Protein-based nanocomposites consisting of wheat gluten matrix (WG) and montmorillonite (MMT) modified with quaternary ammonium salts (QA) were developed by hydroalcoholic solvent casting. A simple clay-modifying method using five QA salts of different molecular lengths composed of 4, 5, 16, 40 and 72 carbon atoms, represented by C 4N, C 5OHN, C 16N, C 40N and C 72N, was used for mineral intercalation. XRD analysis of composites containing Na–MMT, C 16N–MMT, C 40N–MMT, and C 72N–MMT indicated the diffraction peaks corresponding to the interlayer spacing were absent, demonstrating that these particles were significantly dispersed in the WG matrix. Concentration and dispersion of MMT in the WG protein were the main factors affecting on permeability to water vapor P wv through the films. There was a decrease of P wv when either the raw or the modified MMT was added. The more prominent decrease of the P wv was found for dispersed mineral particles, such as C 16N–MMT, C 40N–MMT, and C 72N–MMT.

Transcriptional autoregulation in development

The authors would like to thank Peter Harte and Mark Ptashne for helpful and stimulating comments on the manuscript. Our lab's research is supported by grants from the NIH and NSF. The UNC Developmental Biology NIH training grant provided support to J.C.P.

Interpretation of the Wingless Gradient Requires Signaling-Induced Self-Inhibition

In a classical view of development, a cell can acquire positional information by reading the local concentration of a morphogen independently of its neighbors. Accordingly, in Drosophila, the morphogen Wingless produced in the wing's prospective distal region activates target genes in a dose-dependent fashion to organize the proximodistal pattern. Here, we show that, in parallel, Wingless triggers two nonautonomous inhibitory programs that play an important role in the establishment of positional information. Cells flanking the source of Wingless produce a negative signal (encoded by notum) that inhibits Wingless signaling in nearby cells. Additionally, in response to Wingless, all prospective wing cells produce an unidentified signal that dampens target gene expression in surrounding cells. Thus, cells influence each other's response to Wingless through at least two modes of lateral inhibition. Without lateral inhibition, some cells acquire ectopic fates. Lateral inhibition may be a general mechanism behind the interpretation of morphogen gradients.

Investigating Human and Drosophila Sprinter Function

Within developing tissues, the Wingless (Wg)/Wnt signaling pathway acts as a morphogen, regulating downstream target activation within receiving cells and ensuring proper growth and organization. Our laboratory has identified a novel highly conserved transmembrane protein in Drosophila named Sprinter (Srt) whose specific mechanism of function is unknown, however without it, Wg is retained within secretory cells (1). We hypothesize that Srt functions by supplying a critical activity required for maturation of Wg protein in its route through the secretory pathway. The focus of this research is to begin to characterize Srt function and determine if the mechanism of function is conserved between humans and Drosophila. Compartment localization studies show that the majority of Drosophila and human Srt reside in an undefined secretory compartment and high level Srt expression causes cells to produce processes that appear to contain vesicles of Wg (Figure 1). Furthermore, we have found that Srt exerts its function by direct physical interaction with the Wg protein and that this interaction is conserved across species. These results suggest that Srt is a multifunctional protein that physically interacts with Wg and perhaps promotes cellular morphological changes that support its dissemination from secretory cells. This data also indicates that these functions are conserved across species. Furthermore, these experiments provide the foundation for evaluating Srt function and conservation in developing Drosophila.

The JAK/STAT pathway regulates proximo‐distal patterning in Drosophila

Abstractunpaired, wingless, and decapentaplegic have independent expression domains. unpaired (upd) (green), wingless (Wg) (red) and decapentaplegic (dpp) (blue) do not overlap in a Drosophila third instar antennal disc. In this issue, Ayala‐Camargo et al. show that these factors mutually repress each other's expression and that this repression is critical for the formation of a single proximo‐distal axis per leg or antenna. upd‐Gal4, UAS‐gfp enhancer trap (green); Wg protein (red) and dpp‐lacZ (anti Beta‐galactosidase) (blue). Anterior is to the left and dorsal is up. See Ayala‐Camargo et al., Developmental Dynamics 236:2721‐2730. © 2007 Wiley‐Liss, Inc.

Cell-autonomous, myristyl-independent activity of the Drosophila Wnt/Wingless antagonist Naked cuticle (Nkd)

Robust animal development, tissue homeostasis, and stem cell renewal requires precise control of the Wnt/β-catenin signaling axis. In the embryo of the fruit fly Drosophila melanogaster, the naked cuticle (nkd) gene attenuates signaling by the Wnt ligand Wingless (Wg) during segmentation. nkd mutants have been reported to exhibit abnormalities in wg transcription, Wg protein distribution and/or transport, and the intracellular response to Wg, but the relationship between each alteration and the molecular mechanism of Nkd action remains unclear. In addition, whether Nkd acts in a cell-autonomous or nonautonomous fashion in the embryo is not known. Mammalian Nkd homologs have N-terminal consensus sequences that direct the post-translational addition of a lipophilic myristoyl moiety, but fly and mosquito Nkd, while sharing N-terminal sequence homology, lack a myristoylation consensus sequence. Here we provide evidence that fly Nkd acts cell-autonomously in the embryo, with its N-terminus able to confer unique functional properties and membrane association that cannot be mimicked in vivo by heterologous myristoylation consensus sequences. In conjunction with our recent observation that Nkd requires nuclear localization for function, our data suggest that Nkd acts at more than one subcellular location within signal-receiving cells to attenuate Wg signaling.

Porcupine-mediated lipid-modification regulates the activity and distribution of Wnt proteins in the chick neural tube

A long-term goal of developmental biology is to understand how morphogens establish gradients that promote proper tissue patterning. A number of reports describe the formation of the Wg (Wnt1) gradient in Drosophila and have shown that Porcupine, a predicted membrane-bound O-acyl transferase, is required for the correct distribution of Wg protein. The discovery that Wnts are palmitoylated on a conserved cysteine residue suggests that porcupine activity and Wnt palmitoylation are important for the generation of Wnt gradients. To establish the role of porcupine in Wnt gradient formation in vertebrates, we tested the role of porcupine/Wnt palmitoylation in human embryonic kidney 293T cells and in the chick neural tube. Our results lead us to conclude that: (1) vertebrate Wnt1 and Wnt3a possess at least one additional site for porcupine-mediated lipid-modification; (2) porcupine-mediated lipid-modification of Wnt proteins promotes their activity in 293T cells and in the chick neural tube; and (3) porcupine-mediated lipid-modification reduces the range of activity of Wnt1 and Wnt3a in the chick neural tube. These findings highlight the importance of porcupine-mediated lipid modifications in the formation of vertebrate Wnt activity gradients.