ABSTRACT
Although the spatial and temporal orchestration of early vertebrate embryogenesis is missing from cell culture systems, recent work suggests that many of the same signals affecting neural induction in vertebrate embryos also regulate embryonic stem (ES) cell neurogenesis. One key regulatory mechanism involved in both in vivo and in vitro neural induction is the inhibition of bone morphogenetic protein (BMP) signals. Wnts and Fibroblast Growth Factors represent additional regulatory influences, which may affect the adoption of neural fates through both BMP-dependent and BMP-independent mechanisms. Insights into neural induction in vivo help to guide paradigms for promoting neural differentiation by ES cells. Conversely, insights into the mechanisms by which ES cells adopt neural fates may provide an improved understanding of neural induction in the early embryo.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Embryonic Induction , Embryonic Stem Cells/physiology , Gene Expression Regulation, Developmental , Nervous System Physiological Phenomena , Nervous System/embryology , Animals , Bone Morphogenetic Proteins/antagonists & inhibitors , Bone Morphogenetic Proteins/genetics , Cell Differentiation , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/metabolism , Humans , Nervous System/growth & development , Signal Transduction , Wnt Proteins/genetics , Wnt Proteins/metabolismABSTRACT
Primary cilia are microtubule-based organelles involved in signal transduction and project from the surface of most vertebrate cells. Proteins that can localize to the cilium, for example, Inversin and Bardet-Biedl syndrome (BBS) proteins, are implicated in both beta-catenin-dependent and -independent Wnt signalling. Given that Inversin and BBS proteins are found both at the cilium and elsewhere in the cell, the role of the cilium itself in Wnt signalling is not clear. Using three separate mutations that disrupt ciliogenesis (affecting Kif3a, Ift88 and Ofd1), we show in this study that the primary cilium restricts the activity of the canonical Wnt pathway in mouse embryos, primary fibroblasts, and embryonic stem cells. Interestingly, unciliated cells activate transcription only in response to Wnt stimulation, but do so much more robustly than ciliated cells. Loss of Kif3a, but not other ciliogenic genes, causes constitutive phosphorylation of Dishevelled (Dvl). Blocking the activity of casein kinase I (CKI) reverses this constitutive Dvl phosphorylation and abrogates pathway hyper-responsiveness. These results suggest that Kif3a restrains canonical Wnt signalling both by restricting the CKI-dependent phosphorylation of Dvl and through a separate ciliary mechanism. More generally, these findings reveal that, in contrast to its role in promoting Hedgehog (Hh) signalling, the cilium restrains canonical Wnt signalling.
Subject(s)
Cilia/metabolism , Kinesins/physiology , Signal Transduction/physiology , Wnt Proteins/metabolism , beta Catenin/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Blotting, Western , Casein Kinase I/metabolism , Cells, Cultured , Dishevelled Proteins , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Fluorescent Antibody Technique , Genotype , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Kinesins/genetics , Kinesins/metabolism , Mice , Models, Biological , Phosphoproteins/metabolism , Proteins/genetics , Proteins/metabolism , Proteins/physiology , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Tumor Suppressor Proteins/physiologyABSTRACT
Suppression of the expression of antiangiogenic factors has been closely associated with multiple malignancies. Thrombospondins 1 and 2 are members of a family of angiogenic inhibitors that are regulated by several oncogenes. In this study, we investigate the role of thrombospondins in Ewing's sarcoma and their regulation by EWS/ETS fusion oncoproteins. We show that the EWS/FLI1 fusion suppresses the expression of thrombospondins in both NIH3T3 fibroblasts and Ewing's sarcoma tumor-derived cell lines. This regulation depends on an intact EWS/FLI1 DNA-binding domain and may involve direct interactions between EWS/FLI1 and thrombospondin promoter regions. Forced expression of thrombospondins in Ewing's sarcoma cell lines inhibited the rate of tumor formation in vivo and markedly decreased the number of microvessels present in the tumors. These findings suggest that thrombospondins play a biologically significant role in tumor vascularization in Ewing's sarcoma and suggest potential therapeutic strategies for future therapeutic intervention.
