Investigation of transcriptional regulation of Foxn1 in fetal thymic epithelial progenitor cells

The thymus in mice and humans originates from the third pharyngeal pouch endoderm. This process is divided into early Foxn1-independent stages and later Foxn1-dependent stages. Foxn1 is indispensible for the differentiation of thymic epithelial progenitor cells (TEPCs) as the development of thymus in Foxn1 mutant mice is arrested around E12.5. The transcriptional changes associated with the developmental of the thymus are poorly understood. In particular, the transcriptional regulation of Foxn1 in the developing thymic rudiment has not been definitively identified. Recently, Pax1, Pax9, Tbx1, and E2Fs have been implicated in transcriptional regulation of Foxn1. However, with the exception of E2Fs, evidence regarding their direct involvement in regulating Foxn1 expression is missing. Therefore, the aims of this thesis were to study the transcriptional regulation of Foxn1 through identification of its regulatory regions and studying the transcriptional changes associated with the developing thymus. These aims were addressed through the use of chromatin-immunoprecipitation technique combined with next-generation sequencing and gene expression analyses of the developing TEPCs. The data presented in this thesis identified H3K4me3 and H3K27ac marked Foxn1 promoter and five H3K4me1 and H3K27ac marked putative enhancer regions. The combination of gene expression analyses and transcription factor binding sites within the above regions suggested Ets1, Isl1, Foxc1, Nfia, Nfib, Srf, Foxo1, Nfatc2, Ing4, Foxa2, Hes1, E2Fs, and p53 as candidate transcriptional regulators of Foxn1. Nfatc2 appears also to be a target of Foxn1 that could play an important role in thymus development by regulating a large set of genes. Comparison of wild type and Foxn1 null thymus showed that Foxn1 could act as positive regulator of Pax1 and negative regulator of Gata3 and Eya1, genes important for third pharyngeal pouch development. The comparison of transcriptome of E10.5 and E11.5 third pharyngeal pouch cells and E12.5 TEPCs showed that genes involved in tissue development are downregulated while those involved in antigen presentation, a process important for thymus function, are upregulated during development. These results also demonstrated a decrease in the activity of transcription factor network involving Hox genes and an increase in the activity of a network involving Nfkb, Rela, and Irf genes. Analysis of signalling pathways suggested that the NFκB signalling pathway could be important for thymus development after E12.5 while TGFβ signalling pathway appeared to be detrimental to Foxn1 expression in thymic epithelial cells. Together, I identified several transcription factors that could be involved in transcriptional regulation of Foxn1 in TEPCs, several genes that could be a target of FOXN1, changes in transcription factor network and signalling pathways associated with the developing thymic rudiment

Foxn1 Regulates Lineage Progression in Cortical and Medullary Thymic Epithelial Cells But Is Dispensable for Medullary Sublineage Divergence

The forkhead transcription factor Foxn1 is indispensable for thymus development, but the mechanisms by which it mediates thymic epithelial cell (TEC) development are poorly understood. To examine the cellular and molecular basis of Foxn1 function, we generated a novel and revertible hypomorphic allele of Foxn1. By varying levels of its expression, we identified a number of features of the Foxn1 system. Here we show that Foxn1 is a powerful regulator of TEC differentiation that is required at multiple intermediate stages of TE lineage development in the fetal and adult thymus. We find no evidence for a role for Foxn1 in TEC fate-choice. Rather, we show it is required for stable entry into both the cortical and medullary TEC differentiation programmes and subsequently is needed at increasing dosage for progression through successive differentiation states in both cortical and medullary TEC. We further demonstrate regulation by Foxn1 of a suite of genes with diverse roles in thymus development and/or function, suggesting it acts as a master regulator of the core thymic epithelial programme rather than regulating a particular aspect of TEC biology. Overall, our data establish a genetics-based model of cellular hierarchies in the TE lineage and provide mechanistic insight relating titration of a single transcription factor to control of lineage progression. Our novel revertible hypomorph system may be similarly applied to analyzing other regulators of development

Foxn1 Is Dynamically Regulated in Thymic Epithelial Cells during Embryogenesis and at the Onset of Thymic Involution

Thymus function requires extensive cross-talk between developing T-cells and the thymic epithelium, which consists of cortical and medullary TEC. The transcription factor FOXN1 is the master regulator of TEC differentiation and function, and declining Foxn1 expression with age results in stereotypical thymic involution. Understanding of the dynamics of Foxn1 expression is, however, limited by a lack of single cell resolution data. We have generated a novel reporter of Foxn1 expression, Foxn1G, to monitor changes in Foxn1 expression during embryogenesis and involution. Our data reveal that early differentiation and maturation of cortical and medullary TEC coincides with precise sub-lineage-specific regulation of Foxn1 expression levels. We further show that initiation of thymic involution is associated with reduced cTEC functionality, and proportional expansion of FOXN1-negative TEC in both cortical and medullary sub-lineages. Cortex-specific down-regulation of Foxn1 between 1 and 3 months of age may therefore be a key driver of the early stages of age-related thymic involution

Initial seeding of the embryonic thymus by immune-restricted lympho-myeloid progenitors

The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling. T-IPs did not include multipotent stem cells or molecular evidence of T cell-restricted progenitors. Instead, single-cell molecular and functional analysis demonstrated that most fetal T-IPs expressed genes of and had the potential to develop into lymphoid as well as myeloid components of the immune system. Moreover, studies of embryos deficient in the transcriptional regulator RBPJ demonstrated that canonical Notch signaling was not involved in pre-thymic restriction to the T cell lineage or the migration of T-IPs

ymdatta/SimplyClip: v4.1

<p>Finalized version of the project v4.</p&gt

Prognostic role of Oct4, CD44 and c-Myc in radio-chemo-resistant oral cancer patients and their tumourigenic potential in immunodeficient mice

In the present study, we have investigated the prognostic value of known stem cell-associated molecules such as Oct4, CD44 and c-Myc in patients with oral SCC who had received post-surgery radio- and/or chemotherapy. Immunohistochemistry was performed to analyse the expression of Oct4, CD44 and c-Myc in 87 tumour tissues, and the expression profile obtained was correlated with clinicopathological parameters of the patients with oral cancer. Tumourigenic potential of these molecules was also evaluated by in vivo studies. Our results showed significant correlation of Oct4 (OS, p = 0.003; DFS, p = 0.001) and c-Myc (OS, p = 0.01; DFS, p = 0.03) with overall survival and disease-free survival independently. Furthermore, all the three markers in combinations of two markers each, i.e. Oct4 + CD44 (OS, p = 0.003; DFS, p = 0.001), Oct4 + c-Myc (OS, p = 0.0001; DFS, p = 0.0001), CD44 + c-Myc (OS, p = 0.008; DFS, p = 0.02) and in combinations of three markers each, i.e. Oct4 + CD44 + c-Myc (OS, p = 0.0001; DFS, p = 0.0001) also significantly correlated with overall survival and disease-free survival. Univariate and multivariate analyses further established the independent prognostic value of Oct4. Oct4-, CD44- and c-Myc-enriched populations independently induced sarcomatoid carcinomas whereas primary keratinocytes developed poorly differentiated carcinomas in immunodeficient mice. Oct4 and c-Myc independently as well as in combination with CD44 might be useful for the prediction of local recurrence and poor survival of patients with oral cancer which is the novel finding of this study. Oct4, c-Myc and CD44 can be used to predict local recurrence and the outcome of treatment in oral cancer patients. In addition, these molecules may find use as molecular targets for effective therapy

Changes in gene expression with age.

<p><b>(A-H)</b> Graphs show RT-qPCR analysis of the markers shown in WT mice at 1 month and 3 months. Error bars show SD. (<b>A-G</b>) n = 5, (<b>H</b>) n = 3 independent biological experiments.</p

Dynamic regulation of <i>Foxn1</i> is evident in the early stages of TEC differentiation.

<p><b>(A-D)</b> Flow cytometric analysis of E13.5, E15.5 and E17.5 fetal <i>Foxn1</i>^<i>G/+</i> thymic primordia for the markers shown. Plots show data after gating against Lineage⁺ and on total EpCAM⁺ cells. WT, wild type. <b>(A-C)</b> n = 3, (<b>D</b>) n = 4 independent biological experiments.</p