Embryoid body expressing a Wnt reporter (green) and the Otx2 gene (red)
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Wnt signaling mediates the establishment of polarity in embryoid bodies
Derk ten Berge, Wouter Koole, Christophe Fuerer, Matt Fish, Elif Eroglu
Directed differentiation of embryonic stem (ES) cells is important for their eventual use in therapeutic applications. In vivo, differentiation of cells depends on signals and morphogen gradients that provide instructive and positional cues. Wnt proteins are such signals, and play critical roles during the establishment of embryonic polarity and formation of the primary germ layers. ES cells form descendants of all three germ layers when differentiated as aggregates termed embryoid bodies. We report the establishment of anteroposterior polarity and the formation of a primitive streak-like region in the embryoid body, dependent on local activation of the Wnt pathway. In this region, cells undergo an epithelial-to-mesenchymal transition and differentiate into mesendodermal progenitors. Exogenous Wnt3a protein posteriorizes the embryoid body, resulting in predominantly mesendodermal differentiation. Inhibition of Wnt signaling promotes anterior character and results in neurectodermal differentiation. The activation of Wnt signaling and primitive streak formation requires external signals but is self-reinforcing after initiation. Our findings show that the Wnt pathway mediates the local execution of a gastrulation-like process in the embryoid body, which displays an unexpected degree of self-organization.
Publication: Wnt signaling mediates self-organization and axis formation in embryoid bodies Cell Stem Cell, in press
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Mammary stem cells grown for several passages in cell culture in the presence of Wnt protein have retained normal differentiation properties, when transplanted into a mouse. The cells are labeled with green fluorescent protein (GFP).
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Wnt signaling and mammary stem cells
Arial Zeng
Another example of the critical role of Wnt signals and stem cell behavior comes from the mammary gland. This tissue is known to contain a population of multipotent mammary stem cells. The existence of mammary stem cells was previously established by the fact that the mammary gland can be regenerated by transplantation. Elucidation of the cellular signals that maintain mammary stem cells is of broad interest and will lead to the design of more effective treatments for breast cancer, as mutations of Wnt pathway components have been implicated in this disease. Using reporter mice that visualize Wnt signaling in intact animals, we found that Wnt responsive cells reside in the basal epithelial layer of the ducts coinciding with stem cell locations. In mutant mice which have slightly elevated Wnt signaling, the stem cells have a selective growth advantage when transplanted. When we use purified Wnt protein in mammary epithelium cells culture we found that it promotes the maintenance of the stem cells. These Wnt responsive stem cells are able to regenerate an entire mammary gland in transplantation assays (Figure 5). Our data suggest that normal stem cells in the mammary gland are under the control of the Wnt pathway in the sense that Wnt signals promote self-renewal.
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Expression of WntD in an early Drosophila embryo
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WntD pathway-mediated regulation of NF-kB activity during development and the innate immune response
Mark A. McElwain, Dennis C. Ko, Michael D. Gordon
Signaling pathways such as those regulating Nuclear Factor-kappaB (NF-kB)-like transcription factors control many aspects of embryonic development and adult homeostasis. Often, the same pathway is used repeatedly to accomplish different tasks, raising the question of how these pathways are regulated in different biological contexts. While much is known about the major pathways transduced through NF-kB family members, much is still to be learned about how other signaling mechanisms control NF-kB activity. Recently, we demonstrated that Drosophila WntD regulates embryonic dorsal/ventral axis specification and the adult innate immune response by signaling through the Drosophila NF-kb homolog Dorsal. Identifying the WntD receptor and other downstream components is essential for understanding how the signal is transduced, and how axis specification and the immune response are regulated. We have found several candidate pathway components identified in a suppressor screen for modifiers of a WntD overexpression phenotype
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Expression of a Wnt reporter in embryos and in the lung
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Activation of Wnt Signaling During Adult Lung Injury and Repair
Cati Logan
In the lung, several mutant phenotypes have revealed that the Wnt pathway is required for lung development, but its function in adult tissues is not well understood. Here, we have examined the activation of Wnt signaling in adult lungs using two different Wnt reporter mice, TOP-Gal and BAT-Gal. Both reporter lines reveal Wnt signaling in Clara cells, suggesting that Wnts may play a role in Clara cell formation or maintenance. To test this hypothesis, we stimulated Clara cell formation using a Naphthalene-mediated injury model and followed Wnt reporter activation during lung repair in TOP-Gal mice. Naphthalene administration selectively ablates Clara cells and is followed by Clara cell regeneration. New Clara cells arise from an injury-resistant progenitor population called Variant Clara cells, which that reside at specific locations within the lung and proliferate in response to Clara cell loss. TOP-Gal is expressed in Clara Cells within Neuroepithelial Bodies, which harbor Variant Clara cells. BrdU labeling experiments have shown that TOP-Gal positive Clara cells are proliferative, suggesting that the TOP-Gal reporter may mark progenitors for the bronchiolar epithelium and that Wnt signaling is activated during Clara cell regeneration.
The reporter genes are also expressed in cilliated cells, which remain present following Clara cell ablation, and flatten to cover the denuded basement membrane once Clara cells are lost. This finding suggests that Wnt signaling may additionally regulate ciliated cells during lung injury. Current experiments are aimed at assessing the role of Wnt signaling during lung repair by perturbing this pathway in lungs using both in vivo and in vitro approaches.
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Purification of several Wnt proteins
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Purified Wnt5a Protein Activates or Inhibits beta-catenin-TCF Signaling Depending on Receptor Context
Amanda J. Mikels
In the most well-understood Wnt signaling pathway, Wnt binding to Frizzled receptors induces beta-catenin protein stabilization and entry into the nucleus, where it complexes with T-cell factor/lymphoid enhancer factor transcription factors to affect the transcription of target genes. In addition to the canonical pathway, evidence for several other Wnt signaling pathways has accumulated, in particular for Wnt5a, which has therefore been classified as a noncanonical Wnt family member. To study the alternative mechanisms by which Wnt proteins signal, we purified the Wnt5a protein to homogeneity. We find that purified Wnt5a inhibits Wnt3a protein-induced canonical Wnt signaling in a dose-dependent manner, not by influencing beta-catenin levels but by downregulating beta-catenin induced reporter gene expression. The Wnt5a signal is mediated by the orphan tyrosine kinase Ror2, is pertussis toxin insensitive, and does not influence cellular calcium levels. We show that in addition to its inhibitory function, Wnt5a can also activate beta-catenin signaling in the presence of the appropriate Frizzled receptor, Frizzled 4. Thus, this study shows for the first time that a single Wnt ligand can initiate discrete signaling pathways through the activation of two distinct receptors. Based on these and additional observations, we propose a model wherein receptor context dictates Wnt signaling output. In this model, signaling by different Wnt family members is not intrinsically regulated by the Wnt proteins themselves but by receptor availability.
Current work involves characterizing the Ror2 protein with newly derived monoclonal antibodies.
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