Notch3 Is Dispensable for Thymocyte β-Selection and Notch1-Induced T Cell Leukemogenesis

Article (PDF Available)inPLoS ONE 6(9):e24937 · September 2011with29 Reads
DOI: 10.1371/journal.pone.0024937 · Source: PubMed
Abstract
Notch1 (N1) signaling induced by intrathymic Delta-like (DL) ligands is required for T cell lineage commitment as well as self-renewal during "β-selection" of TCRβ⁺CD4⁻CD8⁻ double negative 3 (DN3) T cell progenitors. However, over-expression of the N1 intracellular domain (ICN1) renders N1 activation ligand-independent and drives leukemic transformation during β-selection. DN3 progenitors also express Notch3 (N3) mRNA, and over-expression of ligand-independent mutant N3 (ICN3) influences β-selection and drives T cell leukemogenesis. However, the importance of ligand-activated N3 in promoting β-selection and ICN1-induced T cell leukemogenesis has not been examined. To address these questions we generated mice lacking functional N3. We confirmed that DN3 progenitors express N3 protein using a N3-specific antibody. Surprisingly however, N3-deficient DN3 thymocytes were not defective in generating DP thymocytes under steady state conditions or in more stringent competition assays. To determine if N3 co-operates with N1 to regulate β-selection, we generated N1;N3 compound mutants. However, N3 deficiency did not exacerbate the competitive defect of N1⁺/⁻ DN3 progenitors, demonstrating that N3 does not compensate for limiting N1 during T cell development. Finally, N3 deficiency did not attenuate T cell leukemogenesis induced by conditional expression of ICN1 in DN3 thymocytes. Importantly, we showed that in contrast to N1, N3 has a low binding affinity for DL4, the most abundant intrathymic DL ligand. Thus, despite the profound effects of ectopic ligand-independent N3 activation on T cell development and leukemogenesis, physiologically activated N3 is dispensable for both processes, likely because N3 interacts poorly with intrathymic DL4.

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    • "Notch1 is a central mediator of both T cell leukemogenesis and T cell development. ICN-target genes such as Myc [60,61,62], Hes1 [47,63], Notch3 [64,65] and IGF1R [66] are critical to T cell development and T-ALL, and Notch signaling directly controls expression of T-cell-lineage specific identity genes such as Tcf7 [67,68] and Bcl11b [69]. Not surprisingly, interfering with the expression of Notch1 target genes disrupts Notch1 programing of developing T-or T-ALL cells. "
    [Show abstract] [Hide abstract] ABSTRACT: Growth factor independent 1 (Gfi1) is a transcriptional repressor originally identified as a gene activated in T-cell leukemias induced by Moloney-murine-leukemia virus infection. Notch1 is a transmembrane receptor that is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL). Gfi1 is an important factor in the initiation and maintenance of lymphoid leukemias and its deficiency significantly impedes Notch dependent initiation of T-ALL in animal models. Here, we show that immature hematopoietic cells require Gfi1 to competently integrate Notch-activated signaling. Notch1 activation coupled with Gfi1 deficiency early in T-lineage specification leads to a dramatic loss of T-cells, whereas activation in later stages leaves development unaffected. In Gfi1 deficient multipotent precursors, Notch activation induces lethality and is cell autonomous. Further, without Gfi1, multipotent progenitors do not maintain Notch1-activated global expression profiles typical for T-lineage precursors. In agreement with this, we find that both lymphoid-primed multipotent progenitors (LMPP) and early T lineage progenitors (ETP) do not properly form or function in Gfi1(-/-) mice. These defects correlate with an inability of Gfi1(-/-) progenitors to activate lymphoid genes, including IL7R, Rag1, Flt3 and Notch1. Our data indicate that Gfi1 is required for hematopoietic precursors to withstand Notch1 activation and to maintain Notch1 dependent transcriptional programming to determine early T-lymphoid lineage identity.
    Full-text · Article · Sep 2013
    • "In contrast, Notch3 expression in the mouse peaks at the DN3 stage (Taghon et al., 2006; Shi et al., 2011), after the onset of  versus  T lineage bifurcation (Ciofani et al., 2006). Although there is clear detectable Notch3 expression at the DN2 stage when  and  T cells start to diverge (Shi et al., 2011), it is unclear if these levels are sufficient to mediate these developmental processes, especially because recent data revealed no obvious defect in mouse  T cell development in the absence of Notch3 (Shi et al., 2011; Suliman et al., 2011). In addition, it is unclear how much Jagged2 protein is expressed by the TECs of the mouse, and in situ RNA expression analysis suggests that DLL4 is more abundantly expressed in the mouse cortex compared with Jagged2 (Heinzel et al., 2007), raising the possibility that Notch3 might not be sufficiently activated to mediate this early developmental T cell choice. "
    [Show abstract] [Hide abstract] ABSTRACT: In humans, high Notch activation promotes γδ T cell development, whereas lower levels promote αβ-lineage differentiation. How these different Notch signals are generated has remained unclear. We show that differential Notch receptor-ligand interactions mediate this process. Whereas Delta-like 4 supports both TCR-αβ and -γδ development, Jagged1 induces mainly αβ-lineage differentiation. In contrast, Jagged2-mediated Notch activation primarily results in γδ T cell development and represses αβ-lineage differentiation by inhibiting TCR-β formation. Consistently, TCR-αβ T cell development is rescued through transduction of a TCR-β transgene. Jagged2 induces the strongest Notch signal through interactions with both Notch1 and Notch3, whereas Delta-like 4 primarily binds Notch1. In agreement, Notch3 is a stronger Notch activator and only supports γδ T cell development, whereas Notch1 is a weaker activator supporting both TCR-αβ and -γδ development. Fetal thymus organ cultures in JAG2-deficient thymic lobes or with Notch3-blocking antibodies confirm the importance of Jagged2/Notch3 signaling in human TCR-γδ differentiation. Our findings reveal that differential Notch receptor-ligand interactions mediate human TCR-αβ and -γδ T cell differentiation and provide a mechanistic insight into the high Notch dependency of human γδ T cell development.
    Full-text · Article · Mar 2013
    • "Importantly, one also has to consider that the additional presence of the Notch3 receptor in these cells may alter the balance of Notch signaling activity. While Delta-Like-4 does not seem to be a good ligand for this receptor in the mouse (Suliman et al., 2011), it remains to be investigated whether Delta-Like-4 and/or Jagged2 can bind and activate Notch3 in humans. This is particularly important since Notch3 has been suggested to be a negative regulator of Notch1 activation (Beatus et al., 1999 ), suggesting that Notch3 activation might be another mechanism to down-modulate Notch1 activity . "
    [Show abstract] [Hide abstract] ABSTRACT: Notch signaling is critical during multiple stages of T cell development in both mouse and human. Evidence has emerged in recent years that this pathway might regulate T-lineage differentiation differently between both species. Here, we review our current understanding of how Notch signaling is activated and used during human T cell development. First, we set the stage by describing the developmental steps that make up human T cell development before describing the expression profiles of Notch receptors, ligands, and target genes during this process. To delineate stage-specific roles for Notch signaling during human T cell development, we subsequently try to interpret the functional Notch studies that have been performed in light of these expression profiles and compare this to its suggested role in the mouse.
    Full-text · Article · Jun 2012
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