Notch signals are required to promote T lineage commitment and development and suppress alternative cell fates in the thymus. Although the Notch activating ligand(s) in the thymus is(are) not known, studies have shown that hematopoietic progenitors are sensitive to Delta-like (DL), but not Jagged (Jag)-type ligands. In Chapter 3, I show that DL-expressing bone marrow stromal cell lines exhibit Notch ligand-independent functional heterogeneity in their capacity to support T cell development in vitro. These findings thus suggest the existence of stromal cell-derived signals that work with Notch to support T cell development. In Chapters 4 and 5, I investigated the ability of Fringe proteins to modulate Notch ligand-receptor interactions and the developmental consequences of these interactions for hematopoetic progenitors. Fringe proteins are glycosyl-transferases that enhance Notch activation by DL ligands and inhibit Notch activation by Jag ligands. In Chapter 4 I show that Lunatic Fringe (Lfng) enhances the strength of DL-mediated Notch activation to drive proliferation and expansion of early thymocytes and that DL4 and DL1 display different potencies to induce Notch-dependent outcomes. In Chapter 5, I demonstrate for the first time in a mammalian system that Lfng and Manic Fringe (Mfng) co-operate to enhance DL-Notch interactions and inhibit Jag-Notch interactions in hematopoietic stem cells. Thus, Lfng and Mfng function together to induce T cell development and inhibit B cell, myeloid and NK cell development. Collectively, these data highlight the importance of Fringe proteins in modulating the strength and specificity of Notch signaling levels during hematopoieisis.

The protein Liquid facets (Lqf) promotes endocytosis at the plasma membrane1. Lqf activity is required for proper Notch signaling, likely through facilitating the endocytosis of Notch ligand by indirectly linking ligand to clathrin. A genetic modifier screen to identify genes that interact with lqf was performed by a previous graduate student. Genes identified in the screen might provide new insights into how Lqf promotes endocytosis or how Notch signaling is regulated. In this work, I performed genetic mapping techniques to identify the genes mutated in each complementation group of the screen. I identified the gene mutated in complementation group 6 as mitochondrial alanyl tRNA synthetase (Aats-ala-m). tRNA synthetases link a tRNA to its cognate amino acid during translation. Mitochondrial tRNA synthetases function in the mitochondria in translation. Aats-ala-m genetically interacts with lqf, suggesting the two genes function in the same pathway. In this work, I also identified chromosomal regions where the genes mutated in complementation groups 1,2, and 9 are located. / text

The process of cell fate determination, which establishes the vastly diverse set of neural cell types found in the central nervous system, remains poorly understood. During retinal development, multipotent retinal progenitor cells generate seven major cell types, including photoreceptors, interneurons, and glia, in an ordered temporal sequence. The behavior of these progenitor cells is influenced by the Notch pathway, a widely utilized signal during embryogenesis which can regulate proliferation and cell fate decisions. To examine the underlying genetic changes that occur when Notch1 is removed from individual retinal cells, microarray analysis of single cells from wild type or Notch1 conditional knockout retinas was performed. Notch1 deficient cells downregulated progenitor and cell cycle marker genes, while robustly upregulating genes associated with rod genesis. Single wild type cells expressed markers of both rod photoreceptors and interneurons, suggesting that these cells were in a transitional state. In order to examine the role of Notch signaling in cell fate specification separate from its role in proliferation, Notch1 was genetically removed specifically from newly postmitotic cells. Notch1 deficient cells preferentially became cone photoreceptors at embryonic stages, and rod photoreceptors at postnatal stages. In both cases, this cell fate change occurred at the expense of the other cell types normally produced at that time. In addition, single cell profiling revealed that Inhibitor of differentiation 1 and 3 genes were robustly downregulated in Notch1 deficient cells. Ectopic expression of these genes during postnatal development in wild type retinas was sufficient to drive production of progenitor/Müller glial cells. Moreover, Id1 and 3 partially rescued the production of Müller glial cells and bipolar cells in the absence of Notch1, even in newly postmitotic cells. We propose that after cell cycle exit, retinal precursor cells transition through a period in which they express marker genes of several different cell types as they commit to a fate, likely endowed by their progenitor cell. Specifically, cells that will become bipolars or Müller glia depend on Id-mediated Notch signaling during this transitional state to take on their respective fates.

The generation of the cytotoxic CD8 T cell response is dependent on the functional outcomes imposed by the intrathymic constraints of differentiation and self-tolerance. Although thymic function can be partly replicated in vitro using OP9-DL1 cell cultures to yield CD8 αβ T cell receptor (TCR)-bearing cells from hematopoietic progenitor cells, a comprehensive and functional assessment of entirely in-vitro generated CD8 T cells derived from bone marrow hematopoietic stem cells (BM-HSCs) has not been established and remains controversial. Here we demonstrate that a phenotypic, molecular, and functional signature of in vitro-derived CD8 T cells is akin to that of ex vivo CD8 T cells. Transfer of in vitro-derived CD8 T cells into syngeneic and immunodeficient host mice showed no graft-versus-host response, while a robust homeostatic proliferation was observed, respectively. These findings, along with a diverse and broad TCR repertoire expressed by the in vitro-derived CD8 T cells, allowed for the successful generation of antigen (Ag)-specific T cells to be obtained from an entirely in vitro-generated CD8 T cell pool, which calls for further tailoring of their use against viral infections or malignancies. Furthermore, I demonstrate that Notch signaling regulates the expression of the cytolytic molecule Granzyme A in CD8+ T cells. This is supported by the inability of Notch-deprived TCR-signaled CD8 T cells to express Granzyme A, while CD8 T cells that received Notch signals readily expressed Granzyme A, suggesting that Notch signaling is a prerequisite for induction of this cytolytic molecule. We further demonstrate that Notch signaling by OP9 cells allows for efficient differentiation of conventional effector CD8 T cells from SAP-/- BM-derived HSCs and restricts differentiation of innate CD8 T cells while allowing for differentiation of IL17-producing CD8 T cells from BM-HSCs isolated from Itk-/-Rlk-/- (DKO) mice. Moreover, we reveal that the process of positive and negative selection in vitro is constrained by peptide-MHC (pMHC) class I expressed by the OP9 cells and disclose that the commitment of DP precursors to the CD8 T cell lineage is facilitated by Notch signaling. Our findings further establish the requirement for Notch receptor-ligand interactions throughout intrathymic T cell differentiation.

The generation of the cytotoxic CD8 T cell response is dependent on the functional outcomes imposed by the intrathymic constraints of differentiation and self-tolerance. Although thymic function can be partly replicated in vitro using OP9-DL1 cell cultures to yield CD8 αβ T cell receptor (TCR)-bearing cells from hematopoietic progenitor cells, a comprehensive and functional assessment of entirely in-vitro generated CD8 T cells derived from bone marrow hematopoietic stem cells (BM-HSCs) has not been established and remains controversial. Here we demonstrate that a phenotypic, molecular, and functional signature of in vitro-derived CD8 T cells is akin to that of ex vivo CD8 T cells. Transfer of in vitro-derived CD8 T cells into syngeneic and immunodeficient host mice showed no graft-versus-host response, while a robust homeostatic proliferation was observed, respectively. These findings, along with a diverse and broad TCR repertoire expressed by the in vitro-derived CD8 T cells, allowed for the successful generation of antigen (Ag)-specific T cells to be obtained from an entirely in vitro-generated CD8 T cell pool, which calls for further tailoring of their use against viral infections or malignancies. Furthermore, I demonstrate that Notch signaling regulates the expression of the cytolytic molecule Granzyme A in CD8+ T cells. This is supported by the inability of Notch-deprived TCR-signaled CD8 T cells to express Granzyme A, while CD8 T cells that received Notch signals readily expressed Granzyme A, suggesting that Notch signaling is a prerequisite for induction of this cytolytic molecule. We further demonstrate that Notch signaling by OP9 cells allows for efficient differentiation of conventional effector CD8 T cells from SAP-/- BM-derived HSCs and restricts differentiation of innate CD8 T cells while allowing for differentiation of IL17-producing CD8 T cells from BM-HSCs isolated from Itk-/-Rlk-/- (DKO) mice. Moreover, we reveal that the process of positive and negative selection in vitro is constrained by peptide-MHC (pMHC) class I expressed by the OP9 cells and disclose that the commitment of DP precursors to the CD8 T cell lineage is facilitated by Notch signaling. Our findings further establish the requirement for Notch receptor-ligand interactions throughout intrathymic T cell differentiation.

abstract: Postnatal skeletal muscle repair is dependent on the tight regulation of an adult stem cell population known as satellite cells. In response to injury, these quiescent cells are activated, proliferate and express skeletal muscle-specific genes. The majority of satellite cells will fuse to damaged fibers or form new muscle fibers, while a subset will return to a quiescent state, where they are available for future rounds of repair. Robust muscle repair is dependent on the signals that regulate the mutually exclusive decisions of differentiation and self-renewal. A likely candidate for regulating this process is NUMB, an inhibitor of Notch signaling pathway that has been shown to asymmetrically localize in daughter cells undergoing cell fate decisions. In order to study the role of this protein in muscle repair, an inducible knockout of Numb was made in mice. Numb deficient muscle had a defective repair response to acute induced damage as characterized by smaller myofibers, increased collagen deposition and infiltration of fibrotic cells. Satellite cells isolated from Numb-deficient mice show decreased proliferation rates. Subsequent analyses of gene expression demonstrated that these cells had an aberrantly up-regulated Myostatin (Mstn), an inhibitor of myoblast proliferation. Further, this defect could be rescued with Mstn specific siRNAs. These data indicate that NUMB is necessary for postnatal muscle repair and early proliferative expansion of satellite cells. We used an evolutionary compatible to examine processes controlling satellite cell fate decisions, primary satellite cell lines were generated from Anolis carolinensis. This green anole lizard is evolutionarily the closet animal to mammals that forms de novo muscle tissue while undergoing tail regeneration. The mechanism of regeneration in anoles and the sources of stem cells for skeletal muscle, cartilage and nerves are poorly understood. Thus, satellite cells were isolated from A. carolinensis and analyzed for their plasticity. Anole satellite cells show increased plasticity as compared to mouse as determined by expression of key markers specific for bone and cartilage without administration of exogenous morphogens. These novel data suggest that satellite cells might contribute to more than muscle in tail regeneration of A. carolinensis. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2012

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22317号 / 医博第4558号 / 新制||医||1040(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 斎藤 通紀, 教授 篠原 隆司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19231号 / 医科博第63号 / 新制||医||科5(附属図書館) / 32230 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 長船 健二, 教授 柳田 素子, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20233号 / 医博第4192号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 松田 文彦, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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