The Notch receptor is an evolutionarily highly conserved transmembrane protein essential to a wide spectrum of cellular systems. Notch is especially important to T-cell development, and its deregulation leads to leukemia. Although not well characterized, Notch signaling continues to play an integral role in peripheral T-cells, in which a unique mode of Notch activation can occur. In contrast to canonical Notch activation initiated by adjacent ligand-expressing cells, T-cell receptor (TCR)-stimulation is sufficient to induce robust Notch signaling. However, the interactions between these two pathways have not been defined. In this dissertation, we show that Notch activation occurs in peripheral T-cells within a few hours post TCR-stimulation and is required for optimal T-cell activation. Utilizing a panel of inhibitors against components of the TCR signaling cascade, we demonstrate that Notch activation is facilitated through initiation of protein kinase C-induced ADAM-metalloprotease activity. Moreover, internalization of Notch via endocytosis is indispensible for this process. Whereas ligand-mediated Notch stimulation relies on mechanical pulling forces that disrupt the autoinhibitory domain of Notch, we hypothesized that in T-cells in the absence of ligands, these conformational changes are induced through chemical adjustments in the endosome, causing alleviation of autoinhibition and receptor activation. Our data show that endocytosis is not only a prerequisite for TCR-induced Notch processing during normal T-cell function, but is essential even in Notch-mutated T-leukemia cells exhibiting constitutively active Notch signaling. Our work has also focused on signaling mechanisms of Notch following receptor activation. The Notch signal is transduced via cleavage of the intracellular portion of the receptor that subsequently translocates to the nucleus where it regulates gene transcription via interactions with its DNA-binding partner, RBPJκ. Utilizing RBPJκ-deficient T-cells, we show that, although Notch signaling is required, RBPJκ-dependent signaling is dispensable for peripheral T-cell proliferation and activation. Using retroviral constructs that encode modified, active forms of Notch restricted to the nucleus or cytoplasm, we provide evidence that Notch signaling may utilize RBPJκ-independent pathways for signal transduction. In conclusion, T-cells have evolved a unique method of Notch receptor activation, described for the first time in this dissertation, as well as novel mechanisms that facilitate downstream signaling.

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

Seven genes involved in metabolism were tested as direct targets of the Notch signalling pathway. For each gene the occupancy of its enhancers by Su(H), its transcriptional response to Notch pathway and its biological functionality was verified in vitro and in vivo.

Notch signalling is an evolutionary highly conserved signalling pathway. It plays various important roles in the regulation of many fundamental cellular processes such as proliferation, stem cell maintenance and differentiation during embryonic and adult development. Notch signalling has a simple transduction pathway. Upon Notch ligand binding to the receptor, the Notch intracellular domain (NICD) is released into the nucleus. The nuclear NICD interacts with the DNA-binding protein Suppressor of Hairless (Su(H)) to activate the expression of target genes, which are silenced by the Su(H)-corepressor complex in the absence of Notch activity. The functions of Notch are very context-dependent, making it important to identify the Notch regulated genes in different processes. Neural stem cells (NSCs) are cells that can divide and differentiate into all kinds of cells within the brain while they self-renew. Notch signalling is one of the key regulators in maintaining NSCs and performs a similar function in both Drosophila and vertebrate NSCs. Drosophila NSCs serve as an ideal model for studying the relationship between Notch function and stem cell behaviours. Although many target genes, such as the Hes genes, have been identified, they cannot fully account for the diversity of Notch responses. Therefore, further functional study of more potential target genes is needed to gain understanding about Notch-regulated NSC maintenance. In this thesis, a group of potential direct Notch target genes are examined for their responsiveness to Notch regulation and their functions in Drosophila NSCs. Previous genome-wide study in the Bray lab has found a number of potential Notch target genes in the Drosophila larval brain, with the characteristics of Notch transcription factor Su(H) binding and mRNA upregulation by Notch over-activation (Zacharioudaki et al. 2016). I first examined the Notch responsive element (NRE) activity of these potential Notch targets and their regulation by Notch both in vivo and in cell lines. The presented findings validated path, cables and Asph as direct Notch target genes in Drosophila NSCs, while syp, lola and Fer2 do not exhibit characteristics of Notch responsive targets in NSCs. The functional roles of two of the responsive genes, path and cables, were subsequently explored in Drosophila larval brains. Firstly, I found that Path, a potential amino acid transporter, is not only important for protecting NSC proliferation under normal and abnormal conditions through integrating growth pathways, but is also required for protecting brain growth under nutrition deprivation. Secondly, the cables gene was connected to a distal NRE through knocking out the suspected NRE region and the gene itself using the CRISPR/Cas9 technique. Subsequent experiments revealed that cables is also required for NSC proliferation. In summary, a group of direct Notch target genes were validated and as a consequence two genes that are important for protecting NSC proliferation were identified.

The differentiation of mesenchymal stem cells (MSCs) to endothelium is a critical but poorly understood feature of tissue vascularisation and considerable scepticism still remains surrounding this important differentiation event. Defining features of endothelial cells (ECs) are their ability to exist as contact-inhibited polarised monolayers that are stabilised by intercellular junctions, and the expression and activity of endothelial markers. During vasculogenesis, communication between MSCs and differentiated ECs or vascular smooth muscle cells, or between MSCs themselves is likely to influence MSC differentiation. In this study, the possibility that cell density can influence MSC differentiation along the EC lineage was examined. High density plating of human bone marrow-derived MSCs induced prominent endothelial characteristics including cobblestone-like morphology, enhanced endothelial networks, acetylated-low density lipoprotein uptake, vascular growth and stimulated expression of characteristic endothelial markers. Mechanistically, this density-dependent process has been defined. Cell-cell contact-induced Notch signalling was a key initiating step regulating commitment towards an EC lineage, whilst VEGF-A stimulation was required to consolidate the EC fate. Thus, this study not only provides evidence that MSC density is an essential microenvironmental factor stimulating the in vitro differentiation of MSCs to ECs but also demonstrates that MSCs can be differentiated to a functional EC. Taken together, defining how these crucial MSC differentiation events are regulated in vitro, provides an insight into how MSCs differentiate to ECs during postnatal neovascularisation and an opportunity for the therapeutic manipulation of MSCs in vivo, enabling targeted modulation of neovascularisation in ischaemia, wound healing and tumourigenesis.

MicroRNA (miRNA) function is required for normal animal development, in particular in stem cell and precursor populations. I hypothesize that miRNAs are similarly required for stem cell maintenance and appropriate fate commitment in the brain. To test the requirement for global microRNA production, I depleted the microRNA biosynthetic enzyme DICER in the developing mouse brain. I found that DICER loss in embryonic neural progenitor cells leads to embryonic lethality with microcephaly. By histological analysis, I found defects in both neural progenitor cell maintenance and cell differentiation. I also identified new candidate microRNAs for this phenotype by profiling miRNAs in DICER-depleted and control cells. Three microRNAs which are good candidates to modulate nervous differentiation are miR-23b, -182, and -34a. I describe the expression pattern and functional characterization of these candidates. In particular, miR-34a depletes neuron production after progenitor cell differentiation in culture, likely by modulating cell cycling and Notch pathway genes.

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

Multiple Sclerosis (MS) is a neurodegenerative autoimmune disease that affects millions of people worldwide. Although the exact cause of MS is unknown, it is clear that CD4+ T helper cells play a significant role, namely T helper 1 (Th1) and T helper 17 (Th17) cells. The Notch family of proteins plays a role in the development and differentiation of T helper cells. Previous data has shown that inhibition of Notch impairs the ability of T helper cell differentiation. Additionally specific inhibition of certain Notch members inhibits specific T helper cell differentiation, for example the inhibition of Notch 1 inhibits Th1 and iTreg polarization [Samon et al., 2008]. However, the effects of the other Notch family members on CD4+ T cells are not fully studied. We propose that Notch 3 plays an extensive role in the regulation of Th1, Th2, Th17, and iTreg polarizations. In addition, we propose that Notch 3 regulates function of T helper cell function in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Data in this thesis show that Notch 3 plays a significant role in the polarization of Th1, Th17 and iTreg polarization [Karlsson et al., 2011]. We present evidence that the heterozygous and homozygous Notch 3 knockout exhibits a significant decrease in polarization toward Th1, Th17 and iTreg cell fates. Exopolysaccharide (EPS) is a compound that has been previously shown to play a protective role in other inflammatory diseases. EPS has been shown to produce anti-inflammatory macrophages. We propose that a similar anti-inflammatory effect might be possible in EAE. We found that EPS had a significant effect on EAE induction, decreasing the onset and peak disease score. EPS also reduced the concentration of IFN-γ, IL17A, and GM-CSF in the supernatants of the splenocytes after restimulation with MOG. Further experimental data is needed to prove the effects of EPS on EAE and the method by which EPS function. These data indicate that Notch 3 could be crucial in regards to EAE due to the effects on Th1 and Th17 which are instrumental in EAE induction [Raphael et al., 2015].

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23424号 / 医科博第129号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 林 康紀, 教授 伊佐 正, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Hearing loss and vestibular dysfunction are inner ear disease states that arise from an array of diverse etiologies that interfere with mechanosensory hair cell function, including: congenital syndromes, noise-induced trauma, ototoxic drugs, and aging. The investigation of normal inner ear development and the pathological aberrations that cause inner ear disease has been previously advanced through formation of an easily generated, scalable, accurate in vitro model system that readily facilitates experimental applications. This model utilizes a 3D floating cell culture protocol which guides differentiation of stem cell aggregates into inner ear organoids, which are vesicles containing a sensory epithelium with functioning mechanosensory hair cells. Inner ear organoid formation enables studying the effects of modulating the signaling pathways that guide developing inner ear structure and function. The Notch signaling pathway heavily influences the formation of the inner ear through two major mechanisms: lateral induction of sensory progenitor cells and lateral inhibition to determine which of those progenitors differentiate into mechanosensory hair cells. The effects of inhibiting Notch signaling within the inner ear organoid system were explored through application of the ɣ-secretase inhibitor MDL28170 (MDL) at a concentration of 25μM on day 8 of organoid culture. Aggregates were harvested on day 32, fixed, sectioned, and stained according to a standard immunohistochemistry protocol. Sections were stained for the mechanosensory hair cell markers Myosin7a (Myo7a) and Sox2. MDL-treated aggregates demonstrated statistically significant reductions in the total number of vesicles and the number of vesicles containing hair cells compared to control aggregates. In contrast to control aggregates which demonstrated two distinct organoid variants (protruding and embedded), MDL-treated aggregates only formed the embedded variant. Differences in the expression pattern of Sox2, which is also a marker of stemness and neural progenitor cells were also noted between the two conditions. MDL-treated aggregates demonstrated regions of ‘ectopic’ Sox2 expression whereas Sox2 expression in control aggregates was consistently expressed within Myo7a+ regions.