Browsing by Subject "Wnt"

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    Investigating neurogenesis and cell type specification in the mammalian thalamus.
    (2012-06) Bluske, Krista K.
    The thalamus mediates a variety of important brain functions that are critical for behavior and survival. A key feature that enables the thalamus to perform such diverse functions is its parcellation into anatomically and functionally distinct groups of neurons called nuclei. The purpose of this project was to identify the origin of neuronal diversity within the thalamus by investigating the process of neurogenesis. During neurogenesis, proliferating progenitor cells begin to divide asymmetrically to generate neurons. The central hypothesis of the research presented herein is that thalamic organization requires the appropriate number and types of neurons to be generated and that these critical processes are regulated during neurogenesis. This work has characterized the different types of progenitor cells present during thalamic neurogenesis. We confirmed the existence of a special population of thalamic progenitor cells, intermediate (or basal) progenitor cells, and identified transcription factors that regulate their formation and/or maintenance. We also addressed the origin of distinct subtypes of neurons. The spatial organization of thalamic progenitor cells into two distinct progenitor domains during neurogenesis is thought to drive the formation of different subtypes of thalamic neurons. Signaling molecules have been proposed to induce the formation of distinct progenitor domains in numerous brain areas, including the thalamus. We provided a detailed characterization of components of the Wnt/β-catenin-mediated transcriptional pathway during thalamic neurogenesis. Based on the pattern of signaling activity, we hypothesized that Wnt/β-catenin-mediated transcription has a function in forming the two progenitor domains during thalamic neurogenesis. Using conditional genetic manipulations of β-catenin, we found that β-catenin-mediated transcription is required for the specification of thalamic progenitor domains. Furthermore, we found that the Wnt/β-catenin signaling pathway functions in parallel with the sonic hedgehog (Shh) signaling pathway, which had been previously shown to specify thalamic progenitor identity in an opposing manner, by independently regulating transcriptional networks in thalamic progenitor cells. Collectively, the process of neurogenesis involves the generation of the correct number of neurons by regulating asymmetric progenitor divisions and generation of appropriate neuronal subtypes through the functions of signaling pathways and transcriptional networks. These mechanisms provide a broad map for the generation and positioning of appropriate types of neurons in the correct locations within the thalamus.
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    A lymphoid enhancer binding factor (Lef) 1 isoform regulates osteoblast maturation.
    (2010-03) Hoeppner, Luke Hilbert
    The canonical Wnt signaling pathway has emerged as an important regulator of bone formation, regeneration and repair. Studies over the past decade have demonstrated that activation of canonical Wnt signaling generally promotes osteoblast proliferation and enhances bone mass, while suppression of Wnt signaling results in bone loss. Gaining a better understanding of Wnt signaling in the context of skeletal metabolism is important because current skeletal regeneration and repair treatments have limitations, anti-resorptive therapies have unknown long-term health consequences and the demand for therapies is rising as our population ages. Osteoporosis is a growing healthcare challenge that affects about 44 million Americans. Cancer survival rates drop drastically in patients suffering from bone metastases. A complete, molecular understanding of the canonical Wnt signaling cascade in the context of skeletal biology will promote the development of new therapies for the treatment of osteoporosis, skeletal cancer metastasis, and other skeletal diseases that result in uncoupling of bone formation and resorption. Lymphoid Enhancer Binding Factor (Lef) 1 is a transcription factor in the canonical Wnt/The canonical Wnt signaling pathway has emerged as an important regulator of bone formation, regeneration and repair. Studies over the past decade have demonstrated that activation of canonical Wnt signaling generally promotes osteoblast proliferation and enhances bone mass, while suppression of Wnt signaling results in bone loss. Gaining a better understanding of Wnt signaling in the context of skeletal metabolism is important because current skeletal regeneration and repair treatments have limitations, anti-resorptive therapies have unknown long-term health consequences and the demand for therapies is rising as our population ages. Osteoporosis is a growing healthcare challenge that affects about 44 million Americans. Cancer survival rates drop drastically in patients suffering from bone metastases. A complete, molecular understanding of the canonical Wnt signaling cascade in the context of skeletal biology will promote the development of new therapies for the treatment of osteoporosis, skeletal cancer metastasis, and other skeletal diseases that result in uncoupling of bone formation and resorption. Lymphoid Enhancer Binding Factor (Lef) 1 is a transcription factor in the canonical Wnt/Lrp5/6/β-catenin signaling cascade, which regulates osteoblast differentiation, bone density and skeletal strength. In this thesis, I describe the expression and function of an alternative Lef1 isoform in osseous cells. Lef1ΔN is a naturally occurring isoform driven by a promoter (p2) within the intron between exons 3 and 4 of Lef1. Lef1ΔN is induced during late osteoblast differentiation. This is opposite to the expression pattern of the full-length Lef1 protein, which as we previously showed, decreases during differentiation. We showed that the Lef1ΔN p2 promoter is active in osteoblasts and Runx2 positively regulates its activity. BMP2 also promotes Lef1ΔN expression, whereas Wnt3a represses it. Lef1ΔN overexpression in differentiating osteoblasts induced osteocalcin and type 1 collagen expression, which suggests Lef1ΔN is a crucial regulator of terminal differentiation in osseous cells. We found Lef1ΔN interacts with β-catenin to activate a Lef1-responsive promoter and stimulate the transcription of genes involved in late osteoblast differentiation. We mapped the region of Lef1ΔN that associates with β-catenin to an element within the first 61 amino acids of Lef1ΔN and showed this region was required to induce type 1 collagen and osteocalcin expression during osteoblast maturation. Taken together, Lef1ΔN interacts with β-catenin to regulate terminal differentiation in osseous cells.
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    Transcriptional regulation of transcription Factor 8
    (2010-05) Broege, Aaron M.
    Zinc-Finger E-box binding protein (ZEB) 1 (Zfhx1a, AREB6, delta-EF1), encoded by the gene tcf8, is a transcription factor that binds to E-box sequences in regulatory regions of its target genes to either repress or activate transcription. ZEB1 induces the epithelial to mesenchymal transition (EMT) during development and cancer progression. The most prominent phenotype of tcf8 null mice is abnormal skeletal development, and ZEB1 inhibits differentiation of mesenchymal precursors into chondrocytes and osteoblasts; however, little is known about the transcriptional regulation of tcf8 throughout these processes. The transcription factor Runx2 is a master regulator of skeletal formation, making it a likely candidate to regulate expression of tcf8 in this tissue. Seven putative Runx binding sites are present in the tcf8 proximal promoter, and we hypothesized that Runx2 regulates tcf8 activity by binding to one or more of these sites. Reporter based assays indicate that Runx2 either represses or activates transcription of tcf8 in a cell type dependent manner and that this activity is mediated through a region between 164 and 112 relative to the translation start site. Furthermore, Runx2 was found to cooperate synergistically with the transcription factor LEF 1 to activate transcription of tcf8 in Cos7 cells. Given that LEF 1 mediates the effects of the canonical Wnt signaling pathway, the effect of the Wnt3a on tcf8 expression was examined in the C2C12 mesenchymal precursor cell line, where an induction of endogenous tcf8 was observed. These data provide the first evidence for regulation of tcf8 by Runx2 and suggest that Runx2 may interact with LEF 1 to mediate the effects of Wnt signaling on expression of tcf8.