[Show abstract][Hide abstract] ABSTRACT: Lunatic Fringe (Lfng) enhances Notch1 activation by Delta-like 4 (DL4) to promote Notch1-dependent T-lineage commitment of thymus-seeding progenitors. Subsequently, Notch1 and T-cell receptor-β (TCRβ)-containing pre-TCR complexes signal CD4/CD8 double-negative 3 (DN3) committed T-cell progenitors to survive, proliferate, and differentiate into CD4/CD8 double-positive (DP) αβ T-cell precursors. Few DP thymocytes develop without Notch1 or pre-TCR signals, whereas ectopic Notch1 activation causes T-cell leukemia. However, mechanisms of a Notch-pre-TCR collaboration during this "β-selection" process are poorly understood. We genetically manipulated Lfng to attenuate or enhance Notch1 activation in DN3 thymocytes without inducing leukemogenesis. We show that Lfng temporally sustains DL-induced Notch1 signaling to prolong proliferative self-renewal of pre-DP thymocytes. Pre-TCR signaling greatly augmented Notch trophic functions to promote robust proliferation of pre-DP progenitors. In contrast, in the absence of DL/Notch signaling, pre-TCR-expressing progenitors rapidly atrophied and differentiated into DP thymocytes. Thus, Lfng prolongs Notch1 signaling to promote self-renewal more than differentiation during the early stages of β-selection. Our data provide novel insights into the Notch-pre-TCR collaboration, and suggest that decreasing Lfng expression during the DN3-DP transition minimizes the potent leukemogenic potential of Notch1 signaling.
[Show abstract][Hide abstract] ABSTRACT: Notch1 activation by Delta-like (DL) Notch ligands is essential to induce T cell commitment and to suppress B cell development from thymus-seeding progenitors. Thymus-seeding progenitor competition for DL4 is critically regulated by Lunatic Fringe (Lfng), which glycosylates epidermal growth factor repeats in the Notch1 extracellular domain to enhance binding avidity for DL ligands. Notch1 activation is also essential for the process of β-selection, which drives TCRβ(+) CD4/CD8 double-negative 3 (DN3) precursors to proliferate and generate a large pool of CD4/CD8 double-positive thymocytes. We have used several genetic approaches to determine the importance of Lfng-Notch1 interactions in regulating competition of preselection and postselection DN3 thymocytes for DL ligands in vivo. Surprisingly, although Lfng overexpression enhanced DL4 binding by preselection DN3a thymocytes, it did not confer them with a competitive advantage in mixed chimeras. In contrast, Lfng overexpression enhanced competition of post-β-selection DN3b precursors for DL ligands. Lfng modification of O-fucose in the Notch1 ligand-binding domain contributed to but was not solely responsible for the developmental effects of Lfng overexpression. Although previous studies have suggested that pre-TCR-deficient DN3 thymocytes compete poorly for DL ligands, Lfng overexpression did not fully restore double-positive thymocyte pools from DN3b cells with pre-TCR signaling defects. Thus, pre-TCR and Notch signaling have largely nonoverlapping functions in β-selection. Collectively, our data reveal that Lfng enhances DN3b precursor competition for intrathymic DL ligands to maximize Notch-induced clonal expansion during the earliest stage of β-selection.
The Journal of Immunology 10/2010; 185(8):4609-17. DOI:10.4049/jimmunol.1002008 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mammalian genomes encode up to four Notch receptors (Notch1-4) and five Notch ligands of the DSL (Delta/Serrate/Lag-2) family, and Notch signaling controls a wide spectrum of developmental processes. Intrathymic Notch1 signaling is essential for several distinct aspects of early T cell development. Notch signaling has also been implicated as a key regulator of peripheral T cell activation and effector cell differentiation, but its functions in these processes remain poorly understood. Notch signaling is dispensable for B cell development in the bone marrow, but it is required to generate the innate-like marginal zone B cell subset in the spleen and may also regulate plasma cell functions. Modification of Notch receptors by fringe glycosyltransferases influences many Notch-dependent aspects of hematopoiesis by altering Notch responsiveness to Delta-like versus Jagged DSL ligands. Here we review recent advances in general aspects of Notch signaling, as well as studies probing Notch functions in these immunological processes.
[Show abstract][Hide abstract] ABSTRACT: Notch2 activation induced by Delta-like-1 (DL1) drives development of splenic marginal zone (MZ) B cells, an innate-like lineage that protects against sepsis. DL1 interacts with Notch2 weakly, but it is not known whether enhancement of DL1-induced Notch2 activation by Fringe glycosyltransferases is important for MZ B cell development. Furthermore, DL1-expressing cells that promote MZ B cell development have not been identified. We show that Lunatic Fringe (Lfng) and Manic Fringe (Mfng) cooperatively enhanced the DL1-Notch2 interaction to promote MZ B cell development. We also identified radio-resistant red pulp endothelial cells in the splenic MZ that express high amounts of DL1 and promoted MZ B generation. Finally, MZ B cell precursor competition for DL1 homeostatically regulated entry into the MZ B cell pool. Our study has revealed that the Fringe-Notch2 interaction has important functions in vivo and provides insights into mechanisms regulating MZ B cell development.
[Show abstract][Hide abstract] ABSTRACT: Notch1 activation regulates T lineage commitment and early T cell development. Fringe glycosyltransferases alter the sensitivity of Notch receptors to Delta-like versus Jagged Notch ligands, but their functions in T lymphopoiesis have not been defined. Here we show that developmental stage-specific expression of the glycosyltransferase lunatic fringe (Lfng) is required for coordination of the access of T cell progenitors to intrathymic niches that support Notch1-dependent phases of T cell development. Lfng-null progenitors generated few thymocytes in competitive assays, whereas Lfng overexpression converted thymocytes into 'supercompetitors' with enhanced binding of Delta-like ligands and blocked T lymphopoiesis from normal progenitors. We suggest that the ability of Lfng and Notch1 to control progenitor competition for limiting cortical niches is an important mechanism for the homeostatic regulation of thymus size.
[Show abstract][Hide abstract] ABSTRACT: Intrathymic Notch1 signaling critically regulates T-lineage specification and commitment as well as T-cell progenitor survival and differentiation. Notch1 activation is continuously required during progression of early CD4/CD8-double-negative thymocytes to the CD4/CD8-double-positive stage. This developmental transition occurs as thymocytes migrate from the corticomedullary junction (CMJ) to the outer subcapsular zone (SCZ) of the thymus. Members of two families of structurally distinct Notch ligands, Delta-like 1 and Jagged-1, are expressed by cortical thymic epithelial cells, but it is not known which ligands are functionally required within the CMJ and SCZ microenvironmental niches. Our laboratory has investigated this question by genetically manipulating thymocyte expression of Lunatic Fringe (L-Fng), a glycosyltransferase that enhances sensitivity of Notch receptors to Delta-like ligands. This approach has revealed that low-threshold intrathymic Notch1 signals instruct multipotent thymus-seeding progenitors to suppress their B-cell potential and choose the T-cell fate. This strategy has also revealed that Delta-like Notch ligands are functionally limiting in both the CMJ and SCZ microenvironmental niches. Finally, we discuss our recent demonstration that L-Fng-mediated competition for Delta-like ligands is an important mechanism for regulating thymus size.
[Show abstract][Hide abstract] ABSTRACT: Signaling through the transmembrane Notch1 receptor directs thymus-seeding progenitors (TSPs) to suppress their B cell potential and 'choose' the T cell fate. Present paradigms suggest that TSPs are contained in the multipotent early T lineage precursor (ETP) subset of thymocytes. However, we show here that the B cell potential of ETPs was not augmented in microenvironments that limited Notch1 activation. Furthermore, low-threshold Notch1 signals suppressed B cell production by TSPs before they reached the ETP stage. Notch1 signals of a higher threshold were needed to drive proliferation of ETPs and development into CD4(+)CD8(+) double-positive thymocytes. Thus, TSPs can be differentiated from all previously identified early T cell progenitors by their robust B cell potential and exquisite sensitivity to Notch1 signals.
[Show abstract][Hide abstract] ABSTRACT: Notch receptors and ligands were first identified in flies and worms, where they were shown to regulate cell proliferation, cell differentiation, and, in particular, binary cell fate decisions in a variety of developmental contexts. The first mammalian Notch homolog was discovered to be a partner in a chromosomal translocation in a subset of human T-cell leukemias. Subsequent studies in mice and humans have shown that Notch signaling plays essential roles at multiple stages of hematopoiesis, and also regulates the development or homeostasis of cells in many tissues and organs. Thus, it is not surprising that mutations which disrupt Notch signaling cause a wide range of cancers and developmental disorders. Perhaps because it is so widely used, Notch signaling is subject to many unusual forms of regulation. In this review, we will first outline key aspects of Notch signaling and its regulation by endocytosis, glycosylation, and ubiquitination. We will then overview recent literature elucidating how Notch regulates cell-lineage decisions in a variety of developmental contexts. Finally, we will describe the roles of dysregulated Notch signaling in causing several types of cancer and other pathologies.