The requirement for Notch signaling at the beta-selection checkpoint in vivo is absolute and independent of the pre-T cell receptor.
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.
Maillard
Ivan
I
Division of Hematology-Oncology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Tu
LiLi
L
Sambandam
Arivazhagan
A
Yashiro-Ohtani
Yumi
Y
Millholland
John
J
Keeshan
Karen
K
Shestova
Olga
O
Xu
Lanwei
L
Bhandoola
Avinash
A
Pear
Warren S
WS
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
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