Browsing by Subject "Forkhead Transcription Factors"
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Item FOXO is the Mediator Linking Temporal Differentiation and the Insulin Signaling Pathway(2005-12-20) Lah, Carol Joonhyun; Cameron, ScottThe timing of differentiation is crucial for the correct development of an organism, because specific pathways can be used reiteratively to differentiate cells. Until recently, the molecular mechanism behind the temporal control of differentiation has remained elusive. Bateman and McNeill (2004) revealed a novel role for the insulin/insulin-like growth factor receptor (InR) pathway in regulating the timing of differentiation in neuronal photoreceptor cells in the Drosophila compound eye. The link between the InR pathway and temporal differentiation is significant, because of the implication that external factors, e.g. nutrition, are tightly coupled to the timing of differentiation. This proposal tests the hypothesis that FOXO, a crucial component of the InR pathway, mediates the regulation of developmental timing. The aims are the following: 1. Observe if dFOXO mutants affect temporal differentiation in the Drosophila eye. 2. Perform epistasis experiments to determine if dFOXO is downstream of other insulin signaling components. 3. Analyze the downstream targets of dFOXO that may play a role in neuronal differentiation.Item Foxo Transcription Factors Control Spermatogonial Stem Cell Self-Renewal And The Initiation Of Spermatogenesis(2013-01-17) Goertz, Meredith Johanna; Castrillon, Diego H., M.D., Ph.D.Spermatogonial stem cells (SSCs), as the foundation for spermatogenesis, must maintain a balance of both self-renewal and differentiation. Although several factors important for these processes have been identified, the fundamental mechanisms regulating SSC self-renewal and differentiation remain essentially unknown. The work presented here describes the discovery of a role for the Foxo forkhead transcription factors in mouse spermatogenesis and SSCs. Foxo1 was found to specifically mark mouse gonocytes, and its cytoplasmic-to-nuclear translocation delineated the gonocyte-to-SSC transition in neonatal testes. In adults, Foxo1 is specifically expressed within a subset of undifferentiated spermatogonia with stem cell potential. Genetic analyses showed that Foxo1 was required for both SSC maintenance as well as the initiation of spermatogenesis, with limited contributions from Foxo3 and Foxo4. Conditional inactivation of PI3K/Akt pathway components in the male germ line confirmed that PI3K signaling regulates Foxo1 stability and subcellular localization, revealing that the Foxos are crucial effectors of PI3K/Akt signaling in SSCs. The nuclear localization of Foxo1, indicating functional activation, was found to correlate with Gfralpha1 expression and thus stem cell potential. Subsequent gene expression analyses identified a complex network of Foxo gene targets that rationalized both the maintenance of SSCs and initiation of differentiation by the Foxos. Taken together, these findings demonstrate that the Foxos, particularly Foxo1, are essential in maintaining the spermatogonial stem cell population, and regulation of the Foxos through the PI3K/Akt signaling pathway is a critical process underlying SSC self-renewal versus differentiation.Item The Role of Foxo Transcription Factors in B Cell Development and Activation(2010-01-12T18:52:39Z) Hinman, Rochelle Marie; Satterhwait, Anne B.A functional immune system depends on a diverse, self tolerant B cell repertoire. Mature B cells distributed throughout secondary lymphoid organs respond to antigenic stimuli by dividing and differentiating into plasma cells and other effector cell types. Signaling from the B cell receptor (BCR) plays a critical role at several points during this developmental process. Cell survival, proliferation, differentiation, death, anergy, and receptor editing may occur in response to BCR stimulation. A variety of factors, including signal strength and duration, cytokine presence, and co-stimulation determine the ultimate B cell fate. In this thesis, the roles Foxo transcription factors play in maintaining B cell homeostasis will be explored. Foxo1, Foxo3, and Foxo4 have both anti-mitogenic and pro-apoptotic properties. The transcription factors are posttranslationally controlled via Akt. When a mature B lymphocyte is stimulated through the BCR, Akt-mediated phosphorylation of Foxos results in their exclusion from the nucleus. In the absence of Foxo nuclear activity, the B cell progresses into the cell cycle. We have discovered a second PI3K-dependent means of control for Foxos, at the level of mRNA expression. Downstream of the BCR, this means of control is unique and functionally relevant. Mature B cells proliferating in response to anti-IgM downregulate Foxo mRNA expression. This is via activation of the PI3K/Btk/BLNK/PLC-gamma2 pathway. Conversely, Foxo mRNA expression is upregulated in immature B cells, both when the tonic/basal signal through the BCR is disrupted and when the BCR is engaged with anti-IgM. Overexpression of Foxo3 mRNA in an immature B cell line promotes anti- IgM induced apoptosis. Primary immature B cells from Foxo3-/- mice have decreased apoptotic response to BCR crosslinking. Thus, at the immature stage of development our work has revealed a potential role for Foxo3 in promoting clonal deletion. Foxo3-/- mice also have reduced frequencies of pre-B and mature recirculating B cells in the blood and bone marrow. The mice demonstrate increased basal levels of IgG2a, IgG3, and IgA. Thus, Foxo3 deficiency affects numerous aspects of B cell development. [Keywords: B Lymphocyte; Foxo transcription factors; B cell receptor (BCR); PI3K; Foxo3; Btk; B cell development; B cell activation]Item The Roles of Forkhead Transcription Factors in Stem Cell and Myogensis(2007-12-17) Alexander, Matthew Scott; Garcia, Joseph A.Vertebrate myogenesis is a highly conserved process that involves the formation, activation, proliferation, and overall regulation of myogenic progenitor cells (MPCs) that are essential for muscle formation, growth, and regeneration following injury. While the process of skeletal muscle development and regeneration has been well-described on a physiological level, the molecular mechanisms that govern the regulation of these cells are poorly understood. Through the utilization of murine transgenic models and gene disruption strategies, I have been able to elucidate important pathways involved in the regulation of MPCs during embryonic myogenesis and adult regeneration following injury. The experiments performed in satisfaction of my dissertation were aimed at defining the biological regulation of Foxk1 and characterization of a novel forkhead factor, Foxj3. Previous studies from our lab have identified the forkhead/winged helix transcription factor, Foxk1, as an essential regulator of MPC activation and quiescence. Firstly, I have undertaken a series of molecular, biochemical, and genetic studies to define the upstream regulation of the Foxk1 gene promoter. Based on evolutionary conservation of the 5' Foxk1 upstream promoter among mouse, rat, and human, I identified a conserved Sox Binding Element (SBE) that I hypothesized as being essential for the transcriptional regulation of Foxk1. I undertook a candidate-based approach, from which I identified Sox15 as being a potent transcriptional activator of Foxk1. Through cell culture, transcriptional assays, electrophorectic mobility shift assays, and transgenic founder analyses, I confirmed that this SBE is essential for the activation of Foxk1 transcription by Sox15. I demonstrated that Sox15 is essential for normal myoblast cell cycle kinetics through siRNA knockdown of endogenous Sox15 and the characterization of the Sox15 mutant mouse model. Finally, I have characterized the novel forkhead transcription factor Foxj3. I have generated a Foxj3 mutant mouse model. I have observed that Foxj3 mutant mice are growth retarded, have impaired skeletal muscle regeneration following injury, and have a significant decrease in the total percentage of Type I oxidative myofibers. When Foxj3 expression is decreased significantly, myoblasts have perturbed cell cycle kinetics and proliferate at a faster rate. Additionally, I demonstrated that Foxj3 is a direct upstream transactivator of Mef2c in skeletal muscle and an essential upstream regulator of the Mef2c-signaling pathway. In conclusion, these studies have elucidated the functional regulation of Foxk1 and its binding partner, Sin3a, in the MPC population. Additionally, I have identified a novel regulator of skeletal muscle myogenesis and myofiber identity, Foxj3, through cell culture assays and generation of the Foxj3 mutant mouse model.Item The Roles of Major Histocompatibility Complex Class I and Foxk1 in Natural Killer Cell Development(2007-12-18) Moody, Leslie Ann; Bennett, MichaelPathways leading to the development of functionally mature Natural Killer cells from bone marrow progenitors are incompletely characterized. Several reports have indicated the necessity of class I Major Histocompatibility Complex-Ly49 interactions to generate functionally mature Natural Killer cells. Natural Killer cells from mice deficient in Major Histocompatibility Complex class I exhibit impaired lytic ability against class I+and class I- targets. It has been proposed that class I interactions with inhibitory Ly49s are required for generation of lytic Natural Killer cells; cells that do not receive these signals fail to become activated. To investigate further the role of class I-Natural Killer cell interactions during development, we produced chimeric mice using class I- mice, in which the hematopoietic system was derived from class I-expressing mice. We discovered that class I+ Natural Killer cells that are developed in a class I- environment are not functional, despite the presence of class I on hematopoietic cells. This indicates that the environment in which Natural Killer cells are developed determines their function and further supports the role of the bone marrow microenvironment in Natural Killer cell development. A complete understanding of Natural Killer cell development would involve determining which transcription factors drive development of Natural Killer cells from stem cells to mature, functional Natural Killer cells. Several transcription factors have been described to be necessary for Natural Killer cell development. Mice lacking these transcription factors often have a deficit in Natural Killer cells in vivo. Here we illustrate a role for the forkhead transcription factor, Foxk1, in Natural Killer cell development. Foxk1-/- mice have significantly fewer Natural Killer cells than do wild-type mice and their remaining Natural Killer cells have decreased cytotoxicity. An increase in the percentage of cells in a developmentally important expansion stage indicates that Foxk1 acts there. However, Foxk1 seems to play no role in the thymic development of Natural Killer cells; cells with phenotypic characteristics of thymus-derived Natural Killer cells are present in Foxk1-/- mice. Our studies show a clear role for Major Histocompatibility Complex class I and Foxk1 in the development of functionally mature Natural Killer cells in mice.