126 research outputs found

    Canonical notch signaling controls the early thymic epithelial progenitor cell state and emergence of the medullary epithelial lineage in fetal thymus development

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    Thymus function depends on the epithelial compartment of the thymic stroma. Cortical thymic epithelial cells (cTECs) regulate T cell lineage commitment and positive selection, while medullary (m) TECs impose central tolerance on the T cell repertoire. During thymus organogenesis, these functionally distinct sub-lineages are thought to arise from a common thymic epithelial progenitor cell (TEPC). However, the mechanisms controlling cTEC and mTEC production from the common TEPC are not understood. Here, we show that emergence of the earliest mTEC lineage-restricted progenitors requires active NOTCH signaling in progenitor TEC and that, once specified, further mTEC development is NOTCH independent. In addition, we demonstrate that persistent NOTCH activity favors maintenance of undifferentiated TEPCs at the expense of cTEC differentiation. Finally, we uncover a cross-regulatory relationship between NOTCH and FOXN1, a master regulator of TEC differentiation. These data establish NOTCH as a potent regulator of TEPC and mTEC fate during fetal thymus development, and are thus of high relevance to strategies aimed at generating/regenerating functional thymic tissue in vitro and in vivo

    Thymic Epithelial Cell Development and Its Dysfunction in Human Diseases

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    Cytokines, Transcription Factors, and the Initiation of T-Cell Development

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    Multipotent blood progenitor cells migrate into the thymus and initiate the T-cell differentiation program. T-cell progenitor cells gradually acquire T-cell characteristics while shedding their multipotentiality for alternative fates. This process is supported by extracellular signaling molecules, including Notch ligands and cytokines, provided by the thymic microenvironment. T-cell development is associated with dynamic change of gene regulatory networks of transcription factors, which interact with these environmental signals. Together with Notch or pre-T-cell-receptor (TCR) signaling, cytokines always control proliferation, survival, and differentiation of early T cells, but little is known regarding their cross talk with transcription factors. However, recent results suggest ways that cytokines expressed in distinct intrathymic niches can specifically modulate key transcription factors. This review discusses how stage-specific roles of cytokines and transcription factors can jointly guide development of early T cells

    DETERMINATION OF THYMIC EPITHELIAL CELL COMPOSITION AND PROLIFERATION DURING THE PERINATAL TO ADULT TRANSITION

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    T-cells develop in the thymus based on signaling from multiple stromal cell types, particularly thymic epithelial cells (TECs). The thymus develops rapidly during the perinatal time period (birth – 10 days in mice) before reaching a period of homeostasis (10 days – 6 weeks). The mechanisms that initially promote and subsequently limit expansion of the TEC compartment are not known. However, previous reports from our lab suggest that the Cyclin D1-RB-E2F pathway plays a key role in regulating the perinatal to adult transition. We have previously shown that inactivation in TEC of retinoblastoma (RB) family members through deletion of RB family members or expression of cyclin D1 maintains perinatal-like TEC proliferation and continued thymus expansion. Although both cortical TEC (cTEC) and medullary TEC (mTEC) are expanded in the K5.D1 thymus, FACs analysis revealed a marked increase in a novel UEA-1int Sca-1- TEC subset, which is not readily classified as belonging to either the cTEC or mTEC lineage. In addition, low level expression of MHC class II and high-level expression of CD24 suggest that the UEA-1int Sca-1- subset contains immature TECs. Cells with this phenotype constitute a small subset of TEC in the wildtype thymus. The K5.D1 UEA-1int Sca-1- subset has a higher proliferative index compared to the wildtype subset

    Towards understanding the signalling requirements of thymic epithelial progenitor cells

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    Thymic epithelial cells (TECs) are indispensable for the development of T cells in the thymus. Two subtypes of TECs exist in the thymus, medullary mTECs and cortical cTECs. Both mTECs and cTECs originate from endodermal thymic epithelial progenitor cells (TEPCs) in the embryo, but how the differentiation of TEPCs is regulated is not well understood. The aims of this thesis were to establish the role of Notch signalling in TEPC differentiation, and how it interacts with known regulators such as FOXN1 and the NFκB pathway. Gene expression data showed that Notch is active in TEPCs and exhibits a correlation with the mTEC lineage. Loss of Notch function led to a significant reduction in the number of mTECs in the thymus, and this can be attributed to aberrant mTEC specification. Furthermore, the duration of Notch activity in determining mTEC number appears limited to the early phase of organogenesis, and precedes RANK/NFκB mediated mTEC proliferation. Gain of Notch function resulted in a considerable shift to a primitive, TEPC-like phenotype, and subsequently a latent increase in mTEC frequency. Finally, transcriptomic and functional analyses pointed to a cross-repressive mechanism between Notch and FOXN1 in TEPCs. Taken together, these results identified Notch as a novel regulator of mTEC specification, likely through maintaining the potency of fetal TEPCs, a prerequisite for mTEC lineage commitment

    Interrogation of transcriptional, regulatory and signalling networks in fetal thymic epithelial cell development via in silico analyses

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    The thymus is the primary lymphoid organ responsible for the development and maturation of T lymphocytes (aka T-cells) in vertebrates. The complex architecture of the thymic microenvironment orchestrates the formation of a diverse and self-tolerant T-cell repertoire capable of supporting the development and maintenance of a functional immune system. The main component of this microenvironment, the thymic epithelium, is crucially required to direct thymus organogenesis and homeostasis, and to mediate T-cell repertoire development and selection. The thymic epithelial progenitor cells (TEPCs) from which the mature thymus develops originate from the endoderm of the 3rd pharyngeal pouch by embryonic day 9 in mouse development (or early week 6 in human embryos). Expression of the transcription factor FOXN1 is required to drive TEPCs differentiation in each thymic epithelial lineage (TEC), while the absence of functional FOXN1 causes athymia. Moreover, forced expression of Foxn1 in mouse embryonic fibroblasts (MEFs) converts these MEFs into TECs that can support the development of a normal thymic system. Despite the great therapeutic potential that TEPCs present in regenerative medicine, there is currently no detailed model describing regulation of the TEPC state and its differentiation into cortical (c) and medullary (m) TECs, or explaining the dominant role of FOXN1 in the thymic epithelial system. Comparative transcriptomics analysis in conjunction with pathway enrichment analysis of the developing TEPCs could reveal the signalling pathways that regulate the early TEPC state and progression into differentiation. Additionally, integrative bioinformatics analysis of transcriptomics and genomics datasets could identify the functional networks that are directly regulated by FOXN1 during early TEC progression. In this thesis I provide, for the first time, an in silico model explaining fetal TEPC differentiation into the functionally distinct TEC lineages, in the cellular, molecular and signalling contexts of thymus early development. Furthermore, I present evidence which suggests that FOXN1 could be a pioneer factor, capable of fully establishing the transcriptional programme that underpins thymic epithelial cell identity and function. Finally, in this thesis, I introduce the development of an interactive thymic-specific database that provides a platform for easy access, analysis and integration of curated bioinformatics datasets

    Epithelial ErbB2 regulation of thymus homeostasis and age-associated T cell mediated immunity

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    The molecular mechanisms governing the functional and structural decline of thymus with age, causing thymic immunosenescence, are incompletely identified. Using a bitransgenic mouse model, Dr Giangreco discovered that the over-expression of receptor tyrosine kinase ErbB2 causes reversible thymic atrophy. The over-expression of epithelial ErbB2 upon doxycycline administration in bitransgenic mice led to decreased thymus size and cellularity, loss of cortical-medullary boundary and abnormal T cell differentiation, bearing similarity to age-dependent thymic involution. This thesis set out to investigate this observation in more detail. I demonstrated that the observed atrophy in bitransgenic thymuses was because of thymus-specific ErbB2 expression, by employing foetal thymic organ cultures. In addition, I showed that over-expression of epithelial ErbB2 disrupted the thymic epithelial cells distribution. Also, an increase in Sca1+Cd49f+ epithelial cells with stem cell potential was noted, explaining why the thymic atrophy in bitransgenic mice was reversible. Exploration of the potential mechanistic pathways found that the thymic atrophy phenotype of K14-NICDER mice, in which epithelial Notch is activated upon tamoxifen administration, resembled the bitransgenic mouse thymic atrophy phenotype. However, mechanistic studies failed to establish ErbB2 acting upstream of Notch, and require further investigation. Administration of Lapatinib, an ErbB2 inhibitor improved the thymic organization and function in aged mice. Lapatinib treatment of aged mice also enhanced vaccine responses to Prevenar 13, a Streptococcus pneumoniae glycoconjugate vaccine, and increased the efficacy of vaccination to protect against subsequent pneumonia challenge. However my results showed that ErbB2 inhibition does not reverse thymic atrophy in scurfy mice, which have truncated Foxp3 protein, and an autoimmune phenotype. In conclusion, this study highlights the importance of ErbB2 in maintaining thymus homeostasis and thymus mediated immunity, and proposes a novel ErbB2 inhibition therapy for rejuvenating an aged thymus, to counter the associated immunosenescence and thereby improve vaccine responses

    Function of Gata-2 in thymic epithelial cells : a transcription factor identified from gene expression analysis of endodermal cells committed to thymic epithelial cell fate

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    The thymus structure composes of clear morphological regions. The T-cell precursors enter the thymus in the cortico-medullary junction and migrate through the cortex towards the sub capsular region and back through to the cortex into the medulla. During this migration process the thymic epithelial cells provide the microenvironment for the maturation and selection of the majority of the peripheral T-cells. The thymic epithelial cells have their origin in the endodermal cells of the ventral aspect of the 3rd pharyngeal pouch while endodermal cells of the dorsal aspect of the 3rd pharyngeal pouch give rise to the parathyroid glands. For a better understanding of genes which might be involved in determination of endodermal cells to the thymic epithelial cell fate, the gene expression profile of the ventral aspect of 3rd pharyngeal pouch was compared to the dorsal aspect of 3rd pharyngeal pouch using microarrays. The analysis revealed 69 genes which were up regulated in the ventral aspect of 3rd pharyngeal pouch. Eleven genes with the largest differential expression values were further assessed (Gata-2, dll-1, C1qdc2, Samd5, Msx2, Msx1, Ehox, Tgfbi, Unc5c, FoxG1, 1110006E14Rik) using RT-PCR and whole mount in situ hybridization. The genes dll-1, Tgfbi, Msx1 and Msx2 are involved in the Notch, Tgf? and Bmp pathways, respectively. All these pathways are associated with thymus development. The role of the genes Ehox, Gata-2, C1qdc2, Samd5 and Unc5c in thymus development is so far undefined. Gata-2, a transcription factor, known to be involved in hematopoiesis, was the only gene of which its expression was detected by gene chip data, RT-PCR and whole mount in situ hybridization. These results identified Gata-2 as a novel candidate that might be involved in the thymic epithelial cell development. To characterize the function of Gata-2 in thymus development, Gata-2 was specifically deleted in thymic epithelial cells using Foxn1-Cre. The thymi of 3, 6, 13, and 25 weeks old mice were removed and detailed studies were performed. FACS analysis of these thymi revealed an increased thymus cellularity in DN1-DN4, CD4, and CD8 in 6 weeks old thymi and onwards. The thymus architecture which was analyzed by H&E and immunohistochemistry (UEA-1, CK8, CK18, ERTR7) was unaffected when Gata-2 was deleted in TECs. The assessment of TEC population of Gata-2 KO mice did not show any difference. But the gene expression analysis of Gata-2 deficient TECs for the genes c-Jun, CXCL-12, CCL-25, IL-7, c-Fos, c-kit ligand, Edn-1, Edn-Ra, und Edn-Rb showed that CXCL-12 and c-kit ligand were higher expressed. CXCL-12 is involved in homing of T-cell precursors while c-kit L is involved in survival and proliferation of T-cell precursors. 7 In conclusion, Gata-2 might negatively regulate the transcription of CXCL-12 and c-kit ligand. A lack of Gata-2 expression in thymic epithelial cells, therefore, might lead to an increased T-cell precursor attraction and survival/proliferation, thus, explaining the higher cellularity observed in thymus of Gata-2 deficient mice
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