Notch signaling augments the canonical Wnt pathway to specify the size of the otic placode

The inner ear derives from a patch of ectoderm defined by expression of the transcription factor Pax2. We recently showed that this Pax2^+ ectoderm gives rise not only to the otic placode but also to the surrounding cranial epidermis, and that Wnt signaling mediates this placode-epidermis fate decision. We now present evidence for reciprocal interactions between the Wnt and Notch signaling pathways during inner ear induction. Activation of Notch1 in Pax2+ ectoderm expands the placodal epithelium at the expense of cranial epidermis, whereas loss of Notch1 leads to a reduction in the size of the otic placode. We show that Wnt signaling positively regulates Notch pathway genes such as Jag1, Notch1 and Hes1, and we have used transgenic Wnt reporter mice to show that Notch signaling can modulate the canonical Wnt pathway. Gain- and loss-of-function mutations in the Notch and Wnt pathways reveal that some aspects of otic placode development - such as Pax8 expression and the morphological thickening of the placode - can be regulated independently by either Notch or Wnt signals. Our results suggest that Wnt signaling specifies the size of the otic placode in two ways, by directly upregulating a subset of otic genes, and by positively regulating components of the Notch signaling pathway, which then act to augment Wnt signaling

Structure-based Discovery of Novel Small Molecule Wnt Signaling Inhibitors by Targeting the Cysteine-rich Domain of Frizzled.

Frizzled is the earliest discovered glycosylated Wnt protein receptor and is critical for the initiation of Wnt signaling. Antagonizing Frizzled is effective in inhibiting the growth of multiple tumor types. The extracellular N terminus of Frizzled contains a conserved cysteine-rich domain that directly interacts with Wnt ligands. Structure-based virtual screening and cell-based assays were used to identify five small molecules that can inhibit canonical Wnt signaling and have low IC50 values in the micromolar range. NMR experiments confirmed that these compounds specifically bind to the Wnt binding site on the Frizzled8 cysteine-rich domain with submicromolar dissociation constants. Our study confirms the feasibility of targeting the Frizzled cysteine-rich domain as an effective way of regulating canonical Wnt signaling. These small molecules can be further optimized into more potent therapeutic agents for regulating abnormal Wnt signaling by targeting Frizzled

Dissecting the dynamics of signaling events in the BMP, WNT, and NODAL cascade during self-organized fate patterning in human gastruloids.

During gastrulation, the pluripotent epiblast self-organizes into the 3 germ layers-endoderm, mesoderm and ectoderm, which eventually form the entire embryo. Decades of research in the mouse embryo have revealed that a signaling cascade involving the Bone Morphogenic Protein (BMP), WNT, and NODAL pathways is necessary for gastrulation. In vivo, WNT and NODAL ligands are expressed near the site of gastrulation in the posterior of the embryo, and knockout of these ligands leads to a failure to gastrulate. These data have led to the prevailing view that a signaling gradient in WNT and NODAL underlies patterning during gastrulation; however, the activities of these pathways in space and time have never been directly observed. In this study, we quantify BMP, WNT, and NODAL signaling dynamics in an in vitro model of human gastrulation. Our data suggest that BMP signaling initiates waves of WNT and NODAL signaling activity that move toward the colony center at a constant rate. Using a simple mathematical model, we show that this wave-like behavior is inconsistent with a reaction-diffusion-based Turing system, indicating that there is no stable signaling gradient of WNT/NODAL. Instead, the final signaling state is homogeneous, and spatial differences arise only from boundary effects. We further show that the durations of WNT and NODAL signaling control mesoderm differentiation, while the duration of BMP signaling controls differentiation of CDX2-positive extra-embryonic cells. The identity of these extra-embryonic cells has been controversial, and we use RNA sequencing (RNA-seq) to obtain their transcriptomes and show that they closely resemble human trophoblast cells in vivo. The domain of BMP signaling is identical to the domain of differentiation of these trophoblast-like cells; however, neither WNT nor NODAL forms a spatial pattern that maps directly to the mesodermal region, suggesting that mesoderm differentiation is controlled dynamically by the combinatorial effect of multiple signals. We synthesize our data into a mathematical model that accurately recapitulates signaling dynamics and predicts cell fate patterning upon chemical and physical perturbations. Taken together, our study shows that the dynamics of signaling events in the BMP, WNT, and NODAL cascade in the absence of a stable signaling gradient control fate patterning of human gastruloids.R01 GM126122 - NIGMS NIH HHSPublished versio

Distinct requirements of wls, wnt9a, wnt5b and gpc4 in regulating chondrocyte maturation and timing of endochondral ossification

Formation of the mandible requires progressive morphologic change, proliferation, differentiation and organization of chondrocytes preceding osteogenesis. The Wnt signaling pathway is involved in regulating bone development and maintenance. Chondrocytes that are fated to become bone require Wnt to polarize and orientate appropriately to initiate the endochondral ossification program. Although the canonical Wnt signaling has been well studied in the context of bone development, the effects of non-canonical Wnt signaling in regulating the timing of cartilage maturation and subsequent bone formation in shaping ventral craniofacial structure is not fully understood.. Here we examined the role of the non-canonical Wnt signaling pathway (wls, gpc4, wnt5b and wnt9a) in regulating zebrafish Meckel's cartilage maturation to the onset of osteogenic differentiation. We found that disruption of wls resulted in a significant loss of craniofacial bone, whereas lack of gpc4, wnt5b and wnt9a resulted in severely delayed endochondral ossification. This study demonstrates the importance of the non-canonical Wnt pathway in regulating coordinated ventral cartilage morphogenesis and ossification

Common activation of canonical Wnt signaling in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is an extremely aggressive malignancy, which carries a dismal prognosis. Activating mutations of the Kras gene are common to the vast majority of human PDA. In addition, recent studies have demonstrated that embryonic signaling pathway such as Hedgehog and Notch are inappropriately upregulated in this disease. The role of another embryonic signaling pathway, namely the canonical Wnt cascade, is still controversial. Here, we use gene array analysis as a platform to demonstrate general activation of the canonical arm of the Wnt pathway in human PDA. Furthermore, we provide evidence for Wnt activation in mouse models of pancreatic cancer. Our results also indicate that Wnt signaling might be activated downstream of Hedgehog signaling, which is an early event in PDA evolution. Wnt inhibition blocked proliferation and induced apoptosis of cultured adenocarcinoma cells, thereby providing evidence to support the development of novel therapeutical strategies for Wnt inhibition in pancreatic adenocarcinoma

Tumor promoter TPA activates Wnt/β-catenin signaling in a casein kinase 1-dependent manner.

The tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) has been defined by its ability to promote tumorigenesis on carcinogen-initiated mouse skin. Activation of Wnt/β-catenin signaling has a decisive role in mouse skin carcinogenesis, but it remains unclear how TPA activates Wnt/β-catenin signaling in mouse skin carcinogenesis. Here, we found that TPA could enhance Wnt/β-catenin signaling in a casein kinase 1 (CK1) ε/δ-dependent manner. TPA stabilized CK1ε and enhanced its kinase activity. TPA further induced the phosphorylation of LRP6 at Thr1479 and Ser1490 and the formation of a CK1ε-LRP6-axin1 complex, leading to an increase in cytosolic β-catenin. Moreover, TPA increased the association of β-catenin with TCF4E in a CK1ε/δ-dependent way, resulting in the activation of Wnt target genes. Consistently, treatment with a selective CK1ε/δ inhibitor SR3029 suppressed TPA-induced skin tumor formation in vivo, probably through blocking Wnt/β-catenin signaling. Taken together, our study has identified a pathway by which TPA activates Wnt/β-catenin signaling

Dissecting the impact of Frizzled receptors in Wnt/beta-catenin signaling of human mesenchymal stem cells

Wnt/beta-catenin signaling is of fundamental importance in the regulation of self-renewal, migration/invasion, and differentiation of human mesenchymal stem cells (hMSCs). Because little information is available about the function of Frizzled receptors (Fzds) as the main receptors of Wnt proteins in hMSCs, we first performed comparative Fzd mRNA expression profiling. Fzd9 and Fzd10 were not expressed in hMSCs. While Fzd3 was expressed at low levels in hMSCs, the other Fzds exhibited high expression rates. Activation and repression of Wnt signaling in hMSCs revealed that the expression levels of Fzd1, Fzd6, and Fzd7 are positively correlated with the Wnt/beta-catenin activation status, whereas Fzd8 exhibited an inverse relation. For studying the functional relevance of Fzds in Wnt/beta-catenin signaling, RNA interference, ectopic expression studies, and rescue approaches were performed in hMSCs carrying a highly sensitive TCF/LEF reporter gene system (Gaussia luciferase). We found that, Fzd1, Fzd5, Fzd7, and Fzd8 are largely involved in Wnt/beta-catenin signaling of hMSCs. Moreover, the knockdown of Fzd5 can be compensated by the ectopic expression of Fzd7. Conversely, the ectopic expression of Fzd5 in Fzd7-knockdown hMSCs resulted in a rescue of Wnt/beta-catenin signaling, pointing to a functional redundancy of Fzd5 and Fzd7

SOST Inhibits Prostate Cancer Invasion.

Inhibitors of Wnt signaling have been shown to be involved in prostate cancer (PC) metastasis; however the role of Sclerostin (Sost) has not yet been explored. Here we show that elevated Wnt signaling derived from Sost deficient osteoblasts promotes PC invasion, while rhSOST has an inhibitory effect. In contrast, rhDKK1 promotes PC elongation and filopodia formation, morphological changes characteristic of an invasive phenotype. Furthermore, rhDKK1 was found to activate canonical Wnt signaling in PC3 cells, suggesting that SOST and DKK1 have opposing roles on Wnt signaling in this context. Gene expression analysis of PC3 cells co-cultured with OBs exhibiting varying amounts of Wnt signaling identified CRIM1 as one of the transcripts upregulated under highly invasive conditions. We found CRIM1 overexpression to also promote cell-invasion. These findings suggest that bone-derived Wnt signaling may enhance PC tropism by promoting CRIM1 expression and facilitating cancer cell invasion and adhesion to bone. We concluded that SOST and DKK1 have opposing effects on PC3 cell invasion and that bone-derived Wnt signaling positively contributes to the invasive phenotypes of PC3 cells by activating CRIM1 expression and facilitating PC-OB physical interaction. As such, we investigated the effects of high concentrations of SOST in vivo. We found that PC3-cells overexpressing SOST injected via the tail vein in NSG mice did not readily metastasize, and those injected intrafemorally had significantly reduced osteolysis, suggesting that targeting the molecular bone environment may influence bone metastatic prognosis in clinical settings