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FIGURE 1
Model of Wnt/β-catenin pathway in the presence and absence of Wnt ligand. This model illustrates the Wnt/β-catenin pathway's activity in response to the presence or absence of the Wnt ligand. Without Wnt, the proteins APC, Axin, CK1, DVL, and GSK3 constitute the destruction complex that controls β-catenin levels, influencing Wnt signaling. Upon Wnt ligand binds with the Frizzled (Fz) receptor and the LRP-6 co-receptor, GSK3 translocates to the membrane and is then internalized into an early endosome and subsequently into multivesicular bodies (MVBs). This process, which sequesters GSK3 and the destruction complex, triggers macropinocytosis in the Wnt pathway. In the cell nucleus, β-catenin forms an active complex with LEF (lymphoid enhancer factor) and TCF (T-cell factor) proteins by displacing TLE/Groucho complexes and recruitment of histone-modifying co-activators such as CBP/p300, BRG1, BCL9, and Pygo. Such a transcriptional switch leads to a change in multiple cellular processes, such as the activation of genes involved in proliferation and cell migration. Additionally, the transmembrane E3 ligases RNF43/ZNRF3 represent important elements down regulators of the Wnt signaling through the ubiquitination and degradation of the Fzd/Lrp5/6 receptor complex via the endolysosomal pathway. While β-catenin is recognized as an important oncogene, it is also pivotal for cell adhesion as a structural component of the cadherin complex structure (detailed in Figure 3) (Figure created with BioRender.com, accessed on 11 July 2023.). APC, adenomatous polyposis coli; GSK3, glycogen synthase kinase 3; LRP-6, LDL-receptor-related protein 6.
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FIGURE 2
Actin cytoskeleton and focal adhesion response to Wnt3a in human corneal stromal fibroblasts. This figure captures the significant changes in the actin cytoskeleton and focal adhesions of human corneal stromal fibroblasts (HCSFs) after a 20-min exposure to Wnt3a. (A) Untreated HCSF shows that the cells adhere normally to the ECM and exhibit less cell movement and abundant focal adhesion sites. (B) Wnt3a treatment leads to a reduction in cell adherence to the ECM, with actin filaments being disassembled and no longer associated with actin, consequently increasing cell motility. The treated cells demonstrated fewer and shorter adhesion sites, a potential factor in the enhanced mobility observed in cancer cells due to the loss of focal adhesions influenced by Wnt signaling. Notably, enlarged lysosomes are observed after Wnt treatment (indicated by the arrow). (C) The diagram illustrates a model for the endocytosis of the focal adhesion upon heightened Wnt signaling. Activation of Wnt signaling (step 1) prompts the macropinocytosis-mediated internalization of Lrp6/Fz/Wnt/GSK3 signalosomes, along with the master regulator of cell adhesion ITGβ1 and other focal adhesion plaques (step 2). This endocytosis process also sequestration of GSK3, and the destruction complex activates the Wnt/β-catenin pathway (Step 3), as per findings reported in Tejeda-Muñoz et al.3 ECM, extracellular matrix; GSK3, glycogen synthase kinase 3; ITGβ1, Integrin beta 1.
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FIGURE 3
Dynamics of integrin-mediated cell-extracellular matrix adhesions and cell migration. This figure delineates the process of integrin-mediated cell adhesion to the extracellular matrix (ECM) and subsequent cell migration, which is crucial for various physiological processes and plays a significant role in cancer metastasis. Focal adhesions (FA), which link the cytoskeleton with the extracellular matrix, are orchestrated through the integrin receptor, mediating bidirectional signaling (detailed in the Zoomed-in panel). The assembly and disassembly of focal adhesions (a key aspect in integrin trafficking) are critical in effective cell migration. As shown in steps 1 to 4 (left panel), cells develop a polarized phenotype and then disassemble FA at the rear position, propelling the cell body forward and forming new adhesion points at the leading edge, coordinating the turnover and creation of adhesions during migration (Figure created with BioRender.com, accessed on 11 July 2023).
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FIGURE 4
Wnt treatment triggers macropinocytosis. (A) The gold standard method to study macropinocytosis is dextran of above 200 nm in diameter. Therefore, the high molecular weight marker tetramethylrhodamine (TMR)-dextran TMR 70 K was tested after Wnt3a protein (100 ng/mL from Peprotech) treatment after 20 min (B), resulting in an increase in the uptake of macropinocytosis. (C) Diagram of macropinocytosis is an actin cytoskeleton-based phenomenon driven by the activation of p21-activated kinase-1 (Pak1) that involves large endocytic vesicles (diameter over 0.2 μm). Its inhibition by amiloride derivatives such as EIPA constitutes the modern method for macropinocytosis. EIPA, ethylisopropyl amiloride.
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FIGURE 5
Genetic expression in dorsal and ventral regions of the early Xenopus laevis gastrula. Genes specifically expressed in the dorsal and ventral fragments of the early Xenopus laevis gastrula have produced a plethora of new genes involved in embryonic patterning. Many components of the dorsal and ventral region of the Spemann organizer in the Xenopus gastrula have been a rich source of new molecules and developmental mechanisms. Many of the novel genes identified encoded secreted antagonists of the BMP and the Wnt pathways. Diagram based and modified from De Robertis and Tejeda-Muñoz.47
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FIGURE 6
There are different ways to activate Wnt signaling, one of them is through GSK3 inhibition by treatment with LiCl or BIO, or CHIR which mimics Wnt signaling. Incubating the Xenopus embryos at the 32-cell stage with the GSK3 inhibitor LiCl (300 mM, panel D) for 7 min or 6-bromoindirubin-3′-oxime (BIO, 30 mM, panel E) and then removing at stage 9.5 and further cultured to the tadpole stage in 0.1 MMR solution, or overnight treatment with CHIR99021 (CHIR, panel F), has very potent phenotypic effects by increasing the early Wnt signal, resulting in an expansion of dorsal-anterior tissues, with a loss of tail structures. Inhibiting V-ATPase (Con A or Baf, panel B and C) results in microcephalic embryos with increased ventral structures, compared with the control embryo in panel A. These indicate that the cell biology of lysosomes, macropinocytosis, membrane trafficking, and ESCRT machinery plays an essential role in vertebrate development and Wnt signaling. (G) The qRT-PCR of blastula stage 9.5 embryos shows that interfering with membrane trafficking with Baf or Con A decreases Wnt target genes Siamois and Xnr3 normalized for ornithine decarboxylase (ODC). An opposite effect is observed with Wnt activators such as LiCl, BIO, and CHIR. (H and I) Example of an in situ hybridization in Xenopus embryos incubated with Baf showing a reduction of the forebrain and midbrain marker otx2 (arrowhead). BIO, 6-bromoindirubin-3-oxime; ESCRT, endosomal sorting complexes required for transport; GSK3, glycogen synthase kinase 3.
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FIGURE 7
Progression of colon cancer from normal crypt to dysregulated signaling. This figure illustrates the progression from a normal intestinal crypt to colorectal cancer (CRC), highlighting the crucial role of APC (adenomatous polyposis coli) mutations in approximately 85% of colon cancer cases. In a normal crypt, a balance of signaling gradients, including BMP, TGF-β, and Wnt, maintain intestinal stem cell functionality and healthy tissue architecture. APC mutations disrupt this equilibrium. Initially, the loss of APC function leads to benign polyps characterized by epithelial dysplasia. As CRC progresses to invasive colon cancer, it typically involves a series of mutations, such as a single activating K-Ras mutation, a homozygous loss of the tumor suppressor Smad4/DPC4 (Deleted in Pancreatic Carcinoma 4), and mutations in both copies p53 genes, amounting to seven mutations on average before malignancy.79-82 These mutations, alongside disruptions in normal signaling gradients, transform normal cells into cancer-associated fibroblasts (CAFs) and colon cancer stem cells, leading to a dysregulated signaling environment conducive to tumor growth. The figure also delineates the rise in Wnt signaling and subsequent decrease in BMP signaling, contributing to increased cancer stemness in the tumor microenvironment. Some CRC cases developed without prior polyposis and are caused by mutations in DNA mismatch repair genes.79, 81-83 Even in these CRC cases, the Wnt pathway is frequently activated, whether through mutations in Axin1, Axin2, RNF43, or ZNRF3, overexpression of R-Spondin 2 or 3, or mutations that stabilize β-catenin (Figure created with BioRender.com, accessed on 11 July 2023).
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Movie S1. Inhibition of Wnt signaling: “no head.” With its fast-embryonic development, large egg size (1–2 mm in diameter), and high numbers of embryos (1500 per female), Xenopus is a great in vivo model for studying vertebrate development and Wnt signaling, and it has been used widely to probe the events in early embryogenesis. The Xenopus model is known to have about 79% of similar genes in common with human disease genes.84 The frog embryo is also a unique system where the dorsal and the ventral sides are easy to distinguish. The difference in maternal pigmentation along the dorsoventral body axis is visible at the 1-cell and early cleavage stage. The ventral side, the darker part, and the light part, the dorsal side where Wnt signaling takes place. The difference in pigmentation is a consequence of a process called cortical rotation. The Wnt pathway is one of the first signals that appear during development.85 Wnt signaling depends completely on β-catenin. In this movie, we can see a control embryo and how dorsal development is taking place. When β-catenin is depleted (with β-catenin morpholino injected with 24 ng four times into the marginal-vegetal region at the two-cell stage), Xenopus embryos lack Spemann organizer dorsal mesoderm and all neural development, resulting in radially ventralized embryos. By tailbud stages, the cement gland, which is a pigmented head structure (inside the white square), an extremely useful anterior marker, is almost fully differentiated.
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