scholarly journals Protein O-fucosyltransferase 1 (Pofut1) regulates lymphoid and myeloid homeostasis through modulation of Notch receptor ligand interactions

Blood ◽  
2011 ◽  
Vol 117 (21) ◽  
pp. 5652-5662 ◽  
Author(s):  
David Yao ◽  
Yuanshuai Huang ◽  
Xiaoran Huang ◽  
Weihuan Wang ◽  
Quanjian Yan ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Notch Receptor ◽  
Cell Surface Expression ◽  
Developmental Defects ◽  
Marginal Zone B Cells ◽  
Notch Ligand ◽  
Notch Receptors ◽  
Ligand Interactions ◽  
Notch Ligands ◽  
T Lymphopoiesis

Abstract Notch signaling is essential for lymphocyte development and is also implicated in myelopoiesis. Notch receptors are modified by O-fucosylation catalyzed by protein O-fucosyltransferase 1 (Pofut1). Fringe enzymes add N-acetylglucosamine to O-fucose and modify Notch signaling by altering the sensitivity of Notch receptors to Notch ligands. To address physiologic functions in hematopoiesis of Notch modified by O-fucose glycans, we examined mice with inducible inactivation of Pofut1 using Mx-Cre. These mice exhibited a reduction in T lymphopoiesis and in the production of marginal-zone B cells, in addition to myeloid hyperplasia. Restoration of Notch1 signaling rescued T lymphopoiesis and the marrow myeloid hyperplasia. After marrow transfer, both cell-autonomous and environmental cues were found to contribute to lymphoid developmental defects and myeloid hyperplasia in Pofut1-deleted mice. Although Pofut1 deficiency slightly decreased cell surface expression of Notch1 and Notch2, it completely abrogated the binding of Notch receptors with Delta-like Notch ligands and suppressed downstream Notch target activation, indicating that O-fucose glycans are critical for efficient Notch-ligand binding that transduce Notch signals. The combined data support a key role for the O-fucose glycans generated by Pofut1 in Notch regulation of hematopoietic homeostasis through modulation of Notch-ligand interactions.

scholarly journals Current Views on the Roles of O-Glycosylation in Controlling Notch-Ligand Interactions

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 309
Author(s):  
Wataru Saiki ◽  
Chenyu Ma ◽  
Tetsuya Okajima ◽  
Hideyuki Takeuchi
Keyword(s):  
Notch Signaling ◽  
100Th Anniversary ◽  
Notch Receptor ◽  
Notch Receptors ◽  
Evolutionarily Conserved ◽  
Future Challenges ◽  
Ligand Interactions ◽  
Epidermal Growth ◽  
Notch Activation ◽  
Human Specific

The 100th anniversary of Notch discovery in Drosophila has recently passed. The Notch is evolutionarily conserved from Drosophila to humans. The discovery of human-specific Notch genes has led to a better understanding of Notch signaling in development and diseases and will continue to stimulate further research in the future. Notch receptors are responsible for cell-to-cell signaling. They are activated by cell-surface ligands located on adjacent cells. Notch activation plays an important role in determining the fate of cells, and dysregulation of Notch signaling results in numerous human diseases. Notch receptors are primarily activated by ligand binding. Many studies in various fields including genetics, developmental biology, biochemistry, and structural biology conducted over the past two decades have revealed that the activation of the Notch receptor is regulated by unique glycan modifications. Such modifications include O-fucose, O-glucose, and O-N-acetylglucosamine (GlcNAc) on epidermal growth factor-like (EGF) repeats located consecutively in the extracellular domain of Notch receptors. Being fine-tuned by glycans is an important property of Notch receptors. In this review article, we summarize the latest findings on the regulation of Notch activation by glycosylation and discuss future challenges.

scholarly journals POS0042 NOTCH 1 INHIBITION INCREASES OSTEOCLAST PROGENITOR ACTIVITY IN THE MOUSE MODEL OF RHEUMATOID ARTHRITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 226.3-226
Author(s):  
M. Filipović ◽  
A. Šućur ◽  
D. Flegar ◽  
Z. Jajić ◽  
M. Ikić Matijašević ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Notch Signaling ◽  
Neutralizing Antibodies ◽  
Osteoclast Differentiation ◽  
Notch Receptor ◽  
Notch 1 ◽  
Collagen Induced Arthritis ◽  
Notch Receptors

Background:Osteoclasts mediate periarticular and systemic bone loss in rheumatoid arthritis (RA). Osteoclast progenitor cells (OCPs) derived from the myeloid lineage are susceptible to regulation through Notch signaling. Murine bone marrow and splenic OCPs, identified as CD45+Ly6G-CD3-B220-NK1.1-CD11blo/+CD115+CCR2+ cells, are specifically increased in arthritis. We previously identified an increased frequency of OCPs expressing Notch receptors in arthritic mice.Objectives:Several studies suggested that Notch signaling modulation affects the course of experimental arthritis. We aimed to determine the effects of Notch receptor signaling inhibition on OCP activity and arthritis severity in murine collagen-induced arthritis (CIA).Methods:Male C57/Bl6 and DBA mice were immunized with chicken type II collagen and treated with i.p. injections of anti-Notch 1 neutralizing antibodies (1mg/kg). Notch receptor 1 through 4 expression on OCPs was analyzed by flow cytometry in periarticular bone marrow (PBM) and spleen (SPL). Gene expression of Notch receptors, ligands and transcription targets as well as osteoclast differentiation genes RANK, cFos and cFms was determined by qPCR from tissues and sorted OCPs. FACS sorted OCPs were stimulated by osteoclastogenic factors (M-CSF and RANKL), in control, IgG, Jagged (Jag)1 or Delta-like (DLL)1 coated wells, with or without anti-Notch 1 antibodies. Research was approved by the Ethics Committee.Results:We confirmed the expression of Notch receptors on OCPs by flow cytometry with Notch 1 and 2 being most abundantly expressed (around 25% and 40% positive OCPs in PBM and 35% and 20% in SPL respectively), with a significant increase of Notch 2 expression in arthritis. Seeding OCPs on DLL1 coated wells significantly increased while seeding on Jag1 coated wells significantly decreased osteoclastogenesis as reflected on the number of TRAP+ osteoclasts and expression of osteoclast differentiation genes. The addition of anti-Notch 1 antibodies to ligand-stimulated OCPs resulted in an increased number of TRAP+ osteoclasts, partially reversing Jag1 inhibition. In vivo treatment with anti-Notch 1 antibodies did not affect total OCP frequency, but increased expression of Notch 4 both in PBM and SPL as seen by flow cytometry and qPCR. Additionally, anti-Notch 1 treatment stimulated Notch transcription factors HES and HEY. Both PBM and SPL cultured OCPs from anti-Notch 1 treated mice produced a higher number of large TRAP+ osteoclasts, doubling the area covered with osteoclasts in the latter compared to untreated mice. Increased osteoclastogenesis in vitro was further confirmed by an increased expression of osteoclast differentiation genes in the treated group.Conclusion:Our results confirm that Notch signaling may represent an important therapeutic target for the regulation of osteoclast activity in arthritis. Both in vitro and in vivo anti-Notch 1 neutralizing antibodies enhanced osteoclastogenesis in CIA model, implying an inhibitory role of Notch 1 signaling in osteoclast differentiation. As Notch 2 expression is increased on OCPs of arthritic mice, we next plan to determine the effects of Notch 2 neutralization on osteoclast activity and arthritis severity.References:[1]Ikić Matijašević M, Flegar D, Kovačić N, Katavić V, Kelava T, Šućur A, et al. Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen-induced arthritis. Clin Exp Immunol. 2016;186(3):321–35.[2]Šućur A, Filipović M, Flegar D, Kelava T, Šisl D, Lukač N, et al. Notch receptors and ligands in inflammatory arthritis – a systematic review. Immunology Letters 2020 Vol. 223, p. 106–14.Acknowledgements:The work has been supported by Croatian Science Foundation projects IP-2018-01-2414, UIP-2017-05-1965 and DOK-2018-09-4276.Disclosure of Interests:None declared.

Notch-Dependent Control of Blood Lineage Development is Modified by Fucosylation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2448-2448
Author(s):  
Lan Zhou ◽  
Quanjian Yan ◽  
David Yao ◽  
Lebing W Li ◽  
Stanton L. Gerson ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Notch Signaling ◽  
Es Cells ◽  
Notch Receptor ◽  
Wild Type ◽  
Notch Ligand ◽  
Hematopoietic Reconstitution ◽  
Myeloid Development

Abstract Notch receptors are conserved cell surface molecules essential for hematopoietic cell fate determination. Activated Notch enhances self-renewal of hematopoietic stem cells and promotes T lymphopoiesis. O-linked fucose moieties attached to the EGF domains of Notch receptors and its modification by Fringe can strongly modulate Notch signaling. Our recently published results indicate that Notch-dependent signaling controls myelopoiesis both in vitro and in vivo, and identify a requirement for Notch fucosylation in the expression of Notch ligand binding activity and Notch signaling efficiency in hematopoietic progenitor cells. In the current study, we tested the hypothesis that fucosylation controlled Notch signaling regulates hematopoietic lineage homeostasis. Genetically-modified mouse embryonic stem (ES) cells deficient in Notch1 receptor (NOTCH1−/−) or pofut1 (POFUT1−/−) that controls O-fucose modification of Notch receptor EGF repeats are studied in an in vitro co-culture assay with Notch ligand-expressing OP9 cells. Activation of Notch in wild type ES cells promotes T lymphopoiesis, while exposure of NOTCH1−/− or POFUT1−/− ES cells to Notch ligand failed to generate T lymphocytes but sustained granulocytic production. When probed with recombinant Notch ligand Dll1 or Dll4, hematopoietic cells derived from wild type ES line displayed robust Notch ligand binding, but cells from NOTCH1−/− or POFUT1−/− ES lines showed completely absent or reduced Notch ligand interaction, respectively. In comparison, ES cells deficient in pofut2 (POFUT2−/−) that controls O-fucose modification on thrombospondin repeats (TSR) displayed a wild type lineage development phenotype and normal Notch ligand binding ability. When examined for their in vivo hematopoietic reconstitution, blood cells derived from NOTCH1−/− or POFUT1−/− ES lines, but not POFUT2−/− ES line, showed enhanced granulocytic but suppressed T and B lymphoid lineage development. These results are consistent with our bone marrow transplantation findings that hematopoietic reconstitution by fucosylation-deficient marrow progenitor cells exhibited increased granulocytopoiesis while wild type or fucosylation-intact marrow cells have normal lineage distribution. Our observations indicate that Notch signaling maintains blood lineage homeostasis by promoting lymphoid lineage development and suppressing overt myeloid development. O-fucose modification of EGF repeats on Notch receptor is essential for this Notch-dependent control of blood lineage homeostasis as deficiency of fucose on Notch receptor results in enhanced myeloid development.

In Vivo Blockade of Individual Notch Ligands and Receptors Provides a New Targeted Therapeutic Approach In Graft-Versus-Host Disease

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 819-819
Author(s):  
Ivy T Tran ◽  
Ashley R Sandy ◽  
Alexis Carulli ◽  
Gloria T Shan ◽  
Vedran Radojcic ◽  
...  
Keyword(s):  
T Cells ◽  
Notch Signaling ◽  
Gamma Secretase ◽  
Notch Receptors ◽  
Alloreactive T Cells ◽  
Donor T Cells ◽  
Notch Inhibition ◽  
Total Body ◽  
Notch Ligands

Abstract Abstract 819 Notch signaling is a cell-cell communication pathway with multiple functions in health and disease. Notch ligands of the Delta-like (Dll1, 3, 4) or Jagged (Jagged1, 2) family interact with one of four mammalian Notch receptors (Notch1-4), leading to proteolytic activation of the receptors by gamma-secretase. We have discovered a critical role for Notch signaling in the differentiation of pathogenic host-reactive T cells during graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT). Expression of the pan-Notch inhibitor DNMAML in donor T cells led to markedly reduced GVHD severity, without causing global immunosuppression (Blood 2011, 117(1): 299–308). These findings identify Notch signaling in alloreactive T cells as an attractive therapeutic target after allo-BMT. To explore preclinical strategies of Notch blockade in GVHD, we first assessed the effects of systemic pan-Notch inhibition with gamma-secretase inhibitors. In the B6 anti-BALB/c MHC-mismatched model of allo-BMT, administration of the gamma-secretase inhibitor dibenzazepine was as efficient as genetic strategies at blocking Notch target gene expression and production of inflammatory cytokines in donor T cells (IFN-γ, TNF-α, IL-2). However, dibenzazepine induced severe gastrointestinal toxicity after total body irradiation due to inhibition of both Notch1 and Notch2 in the gut epithelium. To avoid these side effects, we hypothesized that targeting individual Notch receptors or ligands could provide safe therapeutic Notch blockade after allo-BMT. Among the four mammalian Notch receptors (Notch1-4), donor alloreactive T cells expressed Notch1 and Notch2. Host dendritic cells expressed Notch ligands of the Jagged and Delta-like (Dll) families, with markedly increased Dll4 but not Jagged1/2 transcripts after total body irradiation. This suggested that blockade of Notch1 and/or Notch2 in T cells or Delta-like Notch ligands in dendritic cells could abrogate GVHD. To explore this possibility, we used specific monoclonal antibodies to neutralize Notch receptors and ligands in vivo after allo-BMT (Nature 2006, 444(7122):1083–7; Nature 2010, 464(7291): 1052–7). Combined blockade of Notch1 and Notch2 in vivo reduced the production of key inflammatory cytokines by alloreactive CD4+ and CD8+ T cells to a similar extent as DNMAML-mediated pan-Notch inhibition. Inhibition of Notch1 alone led to a large decrease in cytokine secretion, indicating that Notch1 is a dominant non-redundant Notch receptor in alloreactive T cells. Consistently, transplantation of Notch1-deficient but not Notch2-deficient B6 T cells allowed for decreased GVHD and improved survival in BALB/c recipients, similarly to global Notch inhibition by DNMAML. We then studied the consequences of inhibiting Dll1, Dll4 or both Dll1/Dll4 Notch ligands during acute GVHD. Combined Dll1/Dll4 blockade was as potent as DNMAML expression in decreasing cytokine production by alloreactive T cells, demonstrating that Delta-like and not Jagged ligands are the key Notch agonists at the alloimmune synapse. Dll4 inhibition was superior to Dll1 blockade in reducing cytokine production, abrogating GVHD, and prolonging recipient survival. Importantly, combined Dll1/Dll4 inhibition provided long-term protection against GVHD morbidity and mortality, while avoiding severe gastrointestinal side effects from Notch inhibition. Protection was observed even upon transient Dll1/Dll4 blockade during 1–2 weeks after transplantation. Altogether, our data suggest that Notch1 and Dll4 preferentially interact during alloreactive T cell priming and identify novel strategies to safely and efficiently target individual elements of the Notch pathway after allo-BMT. Humanized antibodies against Notch receptors and ligands were designed to block both mouse and human proteins, thus our preclinical work could lead to new strategies for GVHD control in human patients. Disclosures: Shelton: Genentech Inc.: Employment. Yan:Genentech Inc.: Employment. Siebel:Genentech Inc.: Employment.

scholarly journals Numb regulates the balance between Notch recycling and late-endosome targeting inDrosophilaneural progenitor cells

2016 ◽  
Vol 27 (18) ◽  
pp. 2857-2866 ◽  
Author(s):  
Seth A. Johnson ◽  
Diana Zitserman ◽  
Fabrice Roegiers
Keyword(s):  
Notch Signaling ◽  
Asymmetric Cell Division ◽  
Adaptor Protein ◽  
Notch Signaling Pathway ◽  
Notch Receptor ◽  
Recycling Endosome ◽  
Notch Receptors ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Sensory Organ Precursor

The Notch signaling pathway plays essential roles in both animal development and human disease. Regulation of Notch receptor levels in membrane compartments has been shown to affect signaling in a variety of contexts. Here we used steady-state and pulse-labeling techniques to follow Notch receptors in sensory organ precursor cells in Drosophila. We find that the endosomal adaptor protein Numb regulates levels of Notch receptor trafficking to Rab7-labeled late endosomes but not early endosomes. Using an assay we developed that labels different pools of Notch receptors as they move through the endocytic system, we show that Numb specifically suppresses a recycled Notch receptor subpopulation and that excess Notch signaling in numb mutants requires the recycling endosome GTPase Rab11 activity. Our data therefore suggest that Numb controls the balance between Notch receptor recycling and receptor targeting to late endosomes to regulate signaling output after asymmetric cell division in Drosophila neural progenitors.

scholarly journals Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1

2009 ◽  
Vol 187 (3) ◽  
pp. 343-353 ◽  
Author(s):  
Kazuhide Watanabe ◽  
Tadahiro Nagaoka ◽  
Joseph M. Lee ◽  
Caterina Bianco ◽  
Monica Gonzales ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Notch Receptor ◽  
Carcinoma Cells ◽  
Vertebrate Development ◽  
Notch Receptors ◽  
Epidermal Growth ◽  
Signaling Activation ◽  
Lipid Raft Microdomains ◽  
Golgi Network

Nodal and Notch signaling pathways play essential roles in vertebrate development. Through a yeast two-hybrid screening, we identified Notch3 as a candidate binding partner of the Nodal coreceptor Cripto-1. Coimmunoprecipitation analysis confirmed the binding of Cripto-1 with all four mammalian Notch receptors. Deletion analyses revealed that the binding of Cripto-1 and Notch1 is mediated by the Cripto-1/FRL-1/Cryptic domain of Cripto-1 and the C-terminal region of epidermal growth factor–like repeats of Notch1. Binding of Cripto-1 to Notch1 occurred mainly in the endoplasmic reticulum–Golgi network. Cripto-1 expression resulted in the recruitment of Notch1 protein into lipid raft microdomains and enhancement of the furin-like protein convertase-mediated proteolytic maturation of Notch1 (S1 cleavage). Enhanced S1 cleavage resulted in the sensitization to ligand-induced activation of Notch signaling. In addition, knockdown of Cripto-1 expression in human and mouse embryonal carcinoma cells desensitized the ligand-induced Notch signaling activation. These results suggest a novel role of Cripto-1 in facilitating the posttranslational maturation of Notch receptors.

scholarly journals Notch ligand activity is modulated by glycosphingolipid membrane composition in Drosophila melanogaster

2010 ◽  
Vol 188 (4) ◽  
pp. 581-594 ◽  
Author(s):  
Sophie Hamel ◽  
Jacques Fantini ◽  
François Schweisguth
Keyword(s):  
Protein Interactions ◽  
Receptor Activation ◽  
Notch Receptor ◽  
Binding Motif ◽  
Membrane Composition ◽  
Notch Receptors ◽  
Notch Ligands ◽  
Ligand Activity ◽  
Signaling Activity

Endocytosis of the transmembrane ligands Delta (Dl) and Serrate (Ser) is required for the proper activation of Notch receptors. The E3 ubiquitin ligases Mindbomb1 (Mib1) and Neuralized (Neur) regulate the ubiquitination of Dl and Ser and thereby promote both ligand endocytosis and Notch receptor activation. In this study, we identify the α1,4-N-acetylgalactosaminyltransferase-1 (α4GT1) gene as a gain of function suppressor of Mib1 inhibition. Expression of α4GT1 suppressed the signaling and endocytosis defects of Dl and Ser resulting from the inhibition of mib1 and/or neur activity. Genetic and biochemical evidence indicate that α4GT1 plays a regulatory but nonessential function in Notch signaling via the synthesis of a specific glycosphingolipid (GSL), N5, produced by α4GT1. Furthermore, we show that the extracellular domain of Ser interacts with GSLs in vitro via a conserved GSL-binding motif, raising the possibility that direct GSL–protein interactions modulate the endocytosis of Notch ligands. Together, our data indicate that specific GSLs modulate the signaling activity of Notch ligands.

scholarly journals NOTCH3 signaling is essential for NF-κB activation in TLR-activated macrophages

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Susana López-López ◽  
Eva María Monsalve ◽  
María José Romero de Ávila ◽  
Julia González-Gómez ◽  
Natalia Hernández de León ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Macrophage Activation ◽  
Notch Signaling Pathway ◽  
P38 Map Kinase ◽  
Notch Receptor ◽  
Dependent Manner ◽  
Activated Macrophages ◽  
Notch Receptors ◽  
Inflammatory Processes ◽  
Tlr4 Activation

Abstract Macrophage activation by Toll receptors is an essential event in the development of the response against pathogens. NOTCH signaling pathway is involved in the control of macrophage activation and the inflammatory processes. In this work, we have characterized NOTCH signaling in macrophages activated by Toll-like receptor (TLR) triggering and determined that DLL1 and DLL4 are the main ligands responsible for NOTCH signaling. We have identified ADAM10 as the main protease implicated in NOTCH processing and activation. We have also observed that furin, which processes NOTCH receptors, is induced by TLR signaling in a NOTCH-dependent manner. NOTCH3 is the only NOTCH receptor expressed in resting macrophages. Its expression increased rapidly in the first hours after TLR4 activation, followed by a gradual decrease, which was coincident with an elevation of the expression of the other NOTCH receptors. All NOTCH1, 2 and 3 contribute to the increased NOTCH signaling detected in activated macrophages. We also observed a crosstalk between NOTCH3 and NOTCH1 during macrophage activation. Finally, our results highlight the relevance of NOTCH3 in the activation of NF-κB, increasing p65 phosphorylation by p38 MAP kinase. Our data identify, for the first time, NOTCH3 as a relevant player in the control of inflammation.

scholarly journals Mechanisms in Endocrinology: Notch signaling in skeletal health and disease

2013 ◽  
Vol 168 (6) ◽  
pp. R95-R103 ◽  
Author(s):  
Stefano Zanotti ◽  
Ernesto Canalis
Keyword(s):  
Notch Signaling ◽  
Cell Fate ◽  
Target Genes ◽  
Skeletal Development ◽  
Alagille Syndrome ◽  
Notch Signaling Pathway ◽  
Loss Of Function ◽  
Skeletal Health ◽  
Notch Ligand ◽  
Ligand Interactions

Notch receptors are single-pass transmembrane proteins that determine cell fate. Upon Notch ligand interactions, proteolytic cleavages release the Notch intracellular domain, which translocates to the nucleus to regulate the transcription of target genes, including Hairy enhancer of split (Hes) and Hes related to YRPW motif (Hey). Notch is critical for skeletal development and activity of skeletal cells, and dysregulation of Notch signaling is associated with human diseases affecting the skeleton. Inherited or sporadic mutations in components of the Notch signaling pathway are associated with spondylocostal dysostosis, spondylothoracic dysostosis and recessive brachydactyly, diseases characterized by skeletal patterning defects. Inactivating mutations of the Notch ligandJAG1or ofNOTCH2are associated with Alagille syndrome, and activating mutations inNOTCH2are associated with Hajdu–Cheney syndrome (HCS). Individuals affected by HCS exhibit osteolysis in distal phalanges and osteoporosis. NOTCH is activated in selected tumors, such as osteosarcoma, and in breast cancer cells that form osteolytic bone metastases. In conclusion, Notch regulates skeletal development and bone remodeling, and gain- or loss-of-function mutations of Notch signaling result in important skeletal diseases.

scholarly journals Maintenance of T Cell Specification and Differentiation Requires Recurrent Notch Receptor–Ligand Interactions

2004 ◽  
Vol 200 (4) ◽  
pp. 469-479 ◽  
Author(s):  
Thomas M. Schmitt ◽  
Maria Ciofani ◽  
Howard T. Petrie ◽  
Juan Carlos Zúñiga-Pflücker
Keyword(s):  
T Cell ◽  
Notch Signaling ◽  
Cell Fate ◽  
Stromal Cells ◽  
Cell Lineage ◽  
Notch Receptor ◽  
Lineage Specification ◽  
Cell Specification ◽  
Receptor Ligand ◽  
Ligand Interactions

Notch signaling has been shown to play a pivotal role in inducing T lineage commitment. However, T cell progenitors are known to retain other lineage potential long after the first point at which Notch signaling is required. Thus, additional requirements for Notch signals and the timing of these events relative to intrathymic differentiation remain unknown. Here, we address this issue by culturing subsets of CD4 CD8 double negative (DN) thymocytes on control stromal cells or stromal cells expressing Delta-like 1 (Dll1). All DN subsets were found to require Notch signals to differentiate into CD4+ CD8+ T cells. Using clonal analyses, we show that CD44+ CD25+ (DN2) cells, which appeared committed to the T cell lineage when cultured on Dll1-expressing stromal cells, nonetheless gave rise to natural killer cells with a progenitor frequency similar to that of CD44+ CD25− (DN1) thymocytes when Notch signaling was absent. These data, together with the observation that Dll1 is expressed on stromal cells throughout the thymic cortex, indicates that Notch receptor–ligand interactions are necessary for induction and maintenance of T cell lineage specification at both the DN1 and DN2 stages of T cell development, suggesting that the Notch-induced repression of the B cell fate is temporally separate from Notch-induced commitment to the T lineage.

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