A common bipotent thymocyte precursor gives rise to both lineages of T cells, αβ and γδ. This thesis addresses how the interplay between intrinsic T cell receptor (TCR) signals and cell extrinsic signals provided by Notch and TCR ligands help to assign and support a final lineage fate decision. Emerging data supports a model in which differential TCR signaling capacity plays an instructional role in specifying lineage fate, particularly through induction of the ERK - early growth response gene (Egr) - inhibitor of DNA binding 3 (Id3) pathway. In particular, Id3 expression serves to regulate adoption of the γδ fate. Moreover, Id3 is both necessary and sufficient to enable γδ-lineage cells to differentiate independently of Notch signaling and become competent interferon (IFN)-γ-producing effectors. These findings identify Id3 as a central player that controls both adoption of the γδ fate and their maturation in the thymus. While loss of Notch signaling in γδTCR-expressing CD4-CD8- (DN)3 cells does not affect development, Notch signals are critical for pre-TCR-bearing cells to transition to the CD4+CD8+ (DP) stage of αβ T cell development. Notch signals affect the activation of the PI3K/Akt pathway, which is required for pTα/TCRβ (pre-TCR)-induced survival, differentiation and proliferation of developing αβ-lineage thymocytes. Here, I identify the key molecular players responsible for the interaction between the Notch and PI3K pathways at this critical developmental stage. Notch induction of Hes1 expression is necessary to repress the expression of the PI3K/Akt pathway inhibitor, PTEN, which in turn facilitates pre-TCR-induced differentiation. c-Myc, another critical target of Notch, is required for proliferation during β-selection. Lastly, I find that the majority of DN3 cells expressing both pre-TCR and γδTCR follow the signal strength model for lineage development, and commit and mature along the γδ-lineage. However, manipulation of signal strength, through γδTCR ligand availability or Id3 expression, can skew this development outcome. Taken together, the results from this thesis provide a detailed examination of the molecular mechanisms that are instrumental in determining lineage fate, survival, and proliferation of differentiating thymocytes. Central to these outcomes is the interplay between the Notch, TCR, PI3K, and MAPK signaling pathways.

Thymocyte development is a complex process that requires precise regulation of differentiation and proliferation. Basic helix-loop-helix (bHLH) transcription factors have been shown to be crucial for proper T cell development. HEB and E2A are structurally and functionally related E proteins of the bHLH family. These proteins directly regulate the expression of a number of genes essential for lymphocyte development in a lineage- and stage-specific manner. Abrogation or compromise of their function results in the manifestation of B and T cell developmental defects. Genetic and biochemical studies have provided evidence of a significant degree of functional redundancy among E proteins. The existence of compensational abilities among different E proteins has hampered the investigation and elucidation of E protein function. As such, single gene knockouts demonstrate only limited defects in lymphocyte development. Double E2A-HEB knockouts that could eliminate E protein redundancy are embryonic lethal. In addition, conventional gene knockouts are not well-suited for discerning between intrinsic and extrinsic defects caused by E protein disruption. To eliminate functional compensation and to test the T cell intrinsic roles of E proteins during thymocyte development, we developed a conditional HEB-E2A double knockout. Specifically, we employed a loxP/Lck-Cre recombinase system to drive E protein deletion during early thymocyte development. Using this approach, we were able to reveal overlapping roles for HEB and E2A in thymocyte development that had been obscured in previous single gene knockout studies. We find that simultaneous deletion of HEB and E2A results in a severe block in thymocyte development at the DN to DP stage transition. This developmental block is accompanied by a dramatic decrease in total thymic cellularity, an increase in apoptosis, and a reduction of pTα expression. These developmentally arrested thymocytes exhibit increased proliferation in vivo and dramatic expansion ex vivo in response to IL-7 signaling. Our findings suggest that E2A and HEB are not only critical for the regulation of T cell differentiation but are also necessary to retain developing thymocytes in cell cycle arrest prior to pre-TCR expression. Together, these results imply that E proteins are required to coordinate thymocyte differentiation and proliferation. / Dissertation

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The variations in ER structure and motility under different stages of cell development remain largely unexplored. Here, I observe ER structure and the changes that take place in this structure over time in growing and non-growing live epidermal cells of the pea tendril. The ER was labelled by green fluorescent protein, fused to the KDEL-ER retention signal and confocal scanning laser microscopy was used to localize the fluorescent signal. I found both the structure and motility of growing cells to be different to non-growing cells. The growing cells had a more open arrangement of the cortical ER, fewer lamellae and showed greater tubular dynamics, while the non-growing cells had a denser arrangement of the cortical ER network, with more lamellae and less tubular dynamics. Furthermore, these differences in the cortical ER structure and dynamics were due to growth as, the ER in non-growing cells showed characteristics similar to those seen in growing cells when these cells were induced to grow by the exogenous application of auxin. These changes in ER structure and dynamics were dependant on both the microtubules and actin cytoskeleton networks.

No description available.

A common bipotent thymocyte precursor gives rise to both lineages of T cells, αβ and γδ. This thesis addresses how the interplay between intrinsic T cell receptor (TCR) signals and cell extrinsic signals provided by Notch and TCR ligands help to assign and support a final lineage fate decision. Emerging data supports a model in which differential TCR signaling capacity plays an instructional role in specifying lineage fate, particularly through induction of the ERK - early growth response gene (Egr) - inhibitor of DNA binding 3 (Id3) pathway. In particular, Id3 expression serves to regulate adoption of the γδ fate. Moreover, Id3 is both necessary and sufficient to enable γδ-lineage cells to differentiate independently of Notch signaling and become competent interferon (IFN)-γ-producing effectors. These findings identify Id3 as a central player that controls both adoption of the γδ fate and their maturation in the thymus. While loss of Notch signaling in γδTCR-expressing CD4-CD8- (DN)3 cells does not affect development, Notch signals are critical for pre-TCR-bearing cells to transition to the CD4+CD8+ (DP) stage of αβ T cell development. Notch signals affect the activation of the PI3K/Akt pathway, which is required for pTα/TCRβ (pre-TCR)-induced survival, differentiation and proliferation of developing αβ-lineage thymocytes. Here, I identify the key molecular players responsible for the interaction between the Notch and PI3K pathways at this critical developmental stage. Notch induction of Hes1 expression is necessary to repress the expression of the PI3K/Akt pathway inhibitor, PTEN, which in turn facilitates pre-TCR-induced differentiation. c-Myc, another critical target of Notch, is required for proliferation during β-selection. Lastly, I find that the majority of DN3 cells expressing both pre-TCR and γδTCR follow the signal strength model for lineage development, and commit and mature along the γδ-lineage. However, manipulation of signal strength, through γδTCR ligand availability or Id3 expression, can skew this development outcome. Taken together, the results from this thesis provide a detailed examination of the molecular mechanisms that are instrumental in determining lineage fate, survival, and proliferation of differentiating thymocytes. Central to these outcomes is the interplay between the Notch, TCR, PI3K, and MAPK signaling pathways.

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