title: The requirement for Notch signaling at the beta-selection checkpoint in vivo is absolute and independent of the pre-T cell receptor.
abstract: Genetic inactivation of Notch signaling in CD4(-)CD8(-) double-negative (DN) thymocytes was previously shown to impair T cell receptor (TCR) gene rearrangement and to cause a partial block in CD4(+)CD8(+) double-positive (DP) thymocyte development in mice. In contrast, in vitro cultures suggested that Notch was absolutely required for the generation of DP thymocytes independent of pre-TCR expression and activity. To resolve the respective role of Notch and the pre-TCR, we inhibited Notch-mediated transcriptional activation in vivo with a green fluorescent protein-tagged dominant-negative Mastermind-like 1 (DNMAML) that allowed us to track single cells incapable of Notch signaling. DNMAML expression in DN cells led to decreased production of DP thymocytes but only to a modest decrease in intracellular TCRbeta expression. DNMAML attenuated the pre-TCR-associated increase in cell size and CD27 expression. TCRbeta or TCRalphabeta transgenes failed to rescue DNMAML-related defects. Intrathymic injections of DNMAML(-) or DNMAML(+) DN thymocytes revealed a complete DN/DP transition block, with production of DNMAML(+) DP thymocytes only from cells undergoing late Notch inactivation. These findings indicate that the Notch requirement during the beta-selection checkpoint in vivo is absolute and independent of the pre-TCR, and it depends on transcriptional activation by Notch via the CSL/RBP-J-MAML complex.
authors.affiliation: Division of Hematology-Oncology, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Ivan
authors.initials: I
authors.lastname: Maillard
authors.forename: LiLi
authors.initials: L
authors.lastname: Tu
authors.forename: Arivazhagan
authors.initials: A
authors.lastname: Sambandam
authors.forename: Yumi
authors.initials: Y
authors.lastname: Yashiro-Ohtani
authors.forename: John
authors.initials: J
authors.lastname: Millholland
authors.forename: Karen
authors.initials: K
authors.lastname: Keeshan
authors.forename: Olga
authors.initials: O
authors.lastname: Shestova
authors.forename: Lanwei
authors.initials: L
authors.lastname: Xu
authors.forename: Avinash
authors.initials: A
authors.lastname: Bhandoola
authors.forename: Warren S
authors.initials: WS
authors.lastname: Pear
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000276:immunology*; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers; D005434:Flow Cytometry; D015321:Gene Rearrangement / Q000276:immunology*; D049452:Green Fluorescent Proteins; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000276:immunology; D051880:Receptors, Notch / Q000276:immunology*; D015398:Signal Transduction / Q000276:immunology*; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D013950:Thymus Gland / Q000166:cytology*; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation / Q000276:immunology
chemical_list: D017931:DNA Primers; C486455:Maml1 protein, mouse; D009687:Nuclear Proteins; D011948:Receptors, Antigen, T-Cell; D051880:Receptors, Notch; D014157:Transcription Factors; D049452:Green Fluorescent Proteins