Summary Secreted Wnt morphogens are essential for embryogenesis and homeostasis, and require a lipid/palmitoleoylate modification for receptor binding and activity. Notum is a secreted Wnt antagonist that belongs to the α/β hydrolase superfamily, but its mechanism of action and roles in vertebrate embryogenesis are not fully understood. Here we report that Notum hydrolyzes the Wnt palmitoleoylate adduct extracellularly, resulting in inactivated Wnt proteins that form oxidized oligomers incapable of receptor binding. Thus Notum is a Wnt deacylase, and palmitoleoylation is obligatory for the Wnt structure that maintains its active monomeric conformation. Notum is expressed in naïve ectoderm and neural plate in Xenopus and is required for neural and head induction. These findings suggest that distinct mechanisms of Wnt inactivation by the Tiki protease in the Organizer and the Notum deacylase in presumptive neuroectoderm orchestrate vertebrate brain development.

Glioblastoma multiforme (GBM) is the most common and prognostically unfavorable form of brain tumor. The aggressive and highly invasive phenotype of these tumors makes them among the most anatomically damaging human cancers with a median survival of less than one year. Although canonical WNT pathway activation in cancers has been historically linked to the presence of mutations involving key components of the pathway (APC, β-CATENIN or AXIN proteins), an increasing number of studies suggest that elevated WNT signaling in GBM is initiated by several alternative mechanisms that are involved in different steps of the disease. Therefore, inhibition of WNT signaling may represent a therapeutically relevant approach for GBM treatment. After the selection of a GBM cell model responsive to WNT inhibition, we set out to develop a screening approach for the identification of compounds capable of modulating canonical WNT signaling and associated proliferative responses in GBM cells. Here we show that the small molecule SEN461 inhibits the canonical WNT signaling pathway in GBM cells, with relevant effects at both molecular and phenotypic levels in vitro and in vivo. These include SEN461-induced AXIN stabilization, increased β-CATENIN phosphorylation/degradation, and inhibition of anchorage-independent growth of human GBM cell lines and patient-derived primary tumor cells in vitro. Moreover, in vivo administration of SEN461 antagonized WNT signaling in Xenopus embryos and reduced tumor growth in a GBM xenograft model. These data represent the first demonstration that small molecule-mediated inhibition of WNT signaling may be a potential approach for GBM therapeutics.

Wnt signaling is implicated in a variety of developmental and pathological processes. The molecular mechanisms governing the secretion of Wnt ligands remain to be elucidated. Wntless, an evolutionarily conserved multipass transmembrane protein, is a dedicated secretion factor of Wnt proteins that participates in Drosophila melanogaster embryogenesis. In this study, we show that Xenopus laevis Wntless (XWntless) regulates the secretion of a specific Wnt ligand, XWnt4, and that this regulation is specifically required for eye development in Xenopus. Moreover, the Retromer complex is required for XWntless recycling to regulate the XWnt4-mediated eye development. Inhibition of Retromer function by Vps35 morpholino (MO) resulted in various Wnt deficiency phenotypes, affecting mesoderm induction, gastrulation cell movements, neural induction, neural tube closure, and eye development. Overexpression of XWntless led to the rescue of Vps35 MO-mediated eye defects but not other deficiencies. These results collectively suggest that XWntless and the Retromer complex are required for the efficient secretion of XWnt4, facilitating its role in Xenopus eye development.The Wnt family of glycoproteins comprises one of the largest families of paracrine factors essential for embryonic development and adult tissue homeostasis (reviewed at the Wnt Homepage, http://www.stanford.edu/ϳrnusse/wntwindow .html). It regulates several aspects of biological processes, including cell fate specification, proliferation, migration, and polarity formation (29, 48). The Wnt signaling pathway is initiated by Wnt ligands secreted from Wnt-producing cells. The ligands bind to frizzled receptors and coreceptors expressed on the receiving cells. Wnt ligand perception (5,24,46,49), signaling cascades into receiving cells (8,27), and the consequences of gene expression (17) or cytoskeletal changes (36) are well documented in various contexts. However, relatively limited information is available about the processes in Wnt-producing cells and extracellular spaces. The establishment of the concentration gradient of the Wnt ligand in the extracellular space is mediated by lipoprotein particle formation (33). The Retromer complex is additionally required in Wnt-producing cells and for long-range secretion of Wnt (12). Porcupine is essential for posttranslational modifications, which may be essential for the proper folding and secretion of Wnt ligands (28).Recent achievements in Drosophila melanogaster genetics and genomic RNA interference screening have revealed the existence of a new component of the Wnt secretory pathway, specifically, a dedicated secretion factor of Wg designated Wntless (2), Evi (3), or Sprinter (16). Wntless is an evolutionarily conserved multipass transmembrane protein required solely for Wg secretion. Wntless is not essential for the palmitoylation of Wg, indicating that it does not act on functional Wg production, like Porcupine, another evolutionarily conserved multipass transmembrane protein. Wntless is a regulator of intracellular W...

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Background: The molecular mechanisms governing the formation of the embryonic vascular system remain poorly understood. Here, we show that Disabled-2 (Dab2), a cytosolic adaptor protein, has a pivotal role in the blood vessel formation in Xenopus early embryogenesis.

Dishevelled (Dvl/Dsh) is a key scaffold protein that propagates Wnt signaling essential for embryogenesis and homeostasis. However, whether the antagonism of Wnt signaling that is necessary for vertebrate head formation can be achieved through regulation of Dsh protein stability is unclear. Here, we show that membrane-associated RING-CH2 (March2), a RING-type E3 ubiquitin ligase, antagonizes Wnt signaling by regulating the turnover of Dsh protein via ubiquitin-mediated lysosomal degradation in the prospective head region of We further found that March2 acquires regional and functional specificities for head formation from the Dsh-interacting protein Dapper1 (Dpr1). Dpr1 stabilizes the interaction between March2 and Dsh in order to mediate ubiquitylation and the subsequent degradation of Dsh protein only in the dorso-animal region of embryo. These results suggest that March2 restricts cytosolic pools of Dsh protein and reduces the need for Wnt signaling in precise vertebrate head development.

Phosphotyrosine binding (PTB) domains, which are found in a large number of proteins, have been implicated in signal transduction mediated by growth factor receptors. However, the in vivo roles of these PTB-containing proteins remain to be investigated. Here, we show that Xdpcp (Xenopus dok-PTB containing protein) has a pivotal role in regulating mesendoderm formation in Xenopus, and negatively regulates the activin/nodal signaling pathway. We isolated cDNA for xdpcp and examined its potential role in Xenopus embryogenesis. We found that Xdpcp is strongly expressed in the animal hemisphere at the cleavage and blastula stages. The overexpression of xdpcp RNA affects activin/nodal signaling, which causes defects in mesendoderm formation. In addition, loss of Xdpcp function by injection of morpholino oligonucleotides leads to the expansion of the mesodermal territory. Moreover, we found that axis duplication by ventrally forced expression of activin is recovered by coexpression with Xdpcp. In addition, Xdpcp inhibits the phosphorylation and nuclear translocation of Smad2. Furthermore, we also found that Xdpcp interacts with Alk4, a type I activin receptor, and inhibits activin/nodal signaling by disturbing the interaction between Smad2 and Alk4. Taken together, these results indicate that Xdpcp regulates activin/nodal signaling that is essential for mesendoderm specification. Phosphotyrosine binding (PTB)2 domain-containing proteins are implicated in signal transduction, protein trafficking, and cytoskeletal dynamics (1). The PTB domain is important forprotein-proteininteractionsthatareeitherphosphotyrosinedependent or -independent. As a result, PTB-containing proteins engage in a wide range of cellular functions (2). PTB domain proteins play a pivotal role in signal transduction mediated by various growth factors, including epidermal growth factor (3), insulin (4, 5), nerve growth factor (6), and transforming growth factor-␤ (TGF-␤) (7,8). To mediate TGF-␤ signaling, PTB proteins such as Dab2 (7) and Dok1 (8) act as adaptors to stabilize the interaction between the receptor and Smad2/3. TGF-␤ signaling is implicated in a number of cellular functions such as proliferation, migration, differentiation, and apoptosis. Disturbance of TGF-␤ signaling is related to serious human diseases including cancer, fibrosis, and heritable disorders (9). TGF-␤ signaling is initiated by the binding of ligands to two types of Ser/Thr kinase receptors, called type I and type II receptors. The type I receptor is activated by the type II receptor and propagates intracellular signaling to the nucleus through the phosphorylation of receptor-activated Smads (R-Smad) including Smad1, -2, -3, -5, and -8. Then, the active Smad complex that is formed by the interaction of R-Smad with Smad4 translocates into the nucleus where it regulates the transcription of target genes.TGF-␤ signaling is also crucial for developmental processes including germ-layer specification and patterning during embryogenesis. Xenopus late blastula embryo consists of th...

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