Solanki, ASN; (2017) The role of the transcription factor Gli3 and Hedgehog signalling in fetal B cell and thymocyte development. Doctoral thesis , UCL (University College London).

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Abstract

Gli3 is a Hedgehog (Hh) responsive transcription factor that can function as a transcriptional repressor or activator. Here, we show that Gli3 is required for both fetal B cell and thymocyte development. Gli3 represses Shh in the fetal liver and thymus, hence Gli3 deficiency shows both Gli3 dependent and Gli3 independent Shh dependent changes. Before birth B-cells develop in the fetal liver (FL). Our results show that Gli3 activity in the FL stroma is required for B-cell development. In the Gli3-deficient FL B-cell development is reduced at multiple stages, while the Sonic hedgehog (Shh)-deficient FL shows an increased B-cell development, and Gli3 functions to repress Shh transcription. Using a transgenic Hedgehog (Hh)-reporter mouse, we show that Shh signals directly to developing B-cells, and Hh pathway activation is increased in developing B-cells from the Gli3-deficient FL. RNA-Sequencing confirmed that Hh-mediated transcription was increased in B-lineage cells from Gli3-deficient FL, and showed that these cells expressed reduced levels of B-lineage transcription factors and BCR/pre-BCR-signalling genes. Expression of the master regulators of B-cell development, Ebf1 and Pax5, was reduced in developing B-cells from Gli3-deficient FL and increased in Shh-deficient FL, and in vitro Shh-treatment or neutralisation repressed or induced their expression respectively. In the fetal thymus (FT), Gli3 activity in thymic epithelial cells (TEC) is required for differentiation from CD4+CD8+ to CD4+ single positive (SP4) cell and Gli3 represses Shh. Constitutive deletion of Gli3, and conditional deletion of Gli3 from TEC, reduced differentiation to SP4, whereas conditional deletion of Gli3 from thymocytes did not. Conditional deletion of Shh from TEC increased differentiation and expression of Shh was upregulated in the Gli3-deficient thymus. Use of a transgenic Hh-reporter showed that the Hh pathway was active in thymocytes, and increased in the Gli3-deficient FT. Neutralisation of endogenous Hh proteins in the Gli3-mutant thymus restored SP4 differentiation, indicating that Gli3 in TEC promotes SP4 differentiation by repression of Shh. Transcriptome analysis showed that Hh-mediated transcription was increased but TCR-mediated transcription decreased in Gli3-/- thymocytes.

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