Analysis of Notch1 function by in vitro T cell differentiation of Pax5 mutant lymphoid progenitors.
Signaling through the Notch1 receptor is essential for T cell development in the thymus. Stromal OP9 cells ectopically expressing the Notch ligand Delta-like1 mimic the thymic environment by inducing hemopoietic stem cells to undergo in vitro T cell development. Notch1 is also expressed on Pax5-/- pro-B cells, which are clonable lymphoid progenitors with a latent myeloid potential. In this study, we demonstrate that Pax5-/- progenitors efficiently differentiate in vitro into CD4+CD8+ alphabeta and gammadelta T cells upon coculture with OP9-Delta-like1 cells. In vitro T cell development of Pax5-/- progenitors strictly depends on Notch1 function and progresses through normal developmental stages by expressing T cell markers and rearranging TCRbeta, gamma, and delta loci in the correct temporal sequence. Notch-stimulated Pax5-/- progenitors efficiently down-regulate the expression of B cell-specific genes, consistent with a role of Notch1 in preventing B lymphopoiesis in the thymus. At the same time, Notch signaling rapidly induces cell surface expression of the c-Kit receptor and transcription of the target genes Deltex1 and pre-Talpha concomitant with the activation of TCR Vbeta germline transcription and the regulatory genes GATA3 and Tcf1. These data suggest that Notch1 acts upstream of GATA3 and Tcf1 in early T cell development and regulates Vbeta-DJbeta rearrangements by controlling the chromatin accessibility of Vbeta genes at the TCRbeta locus.
Höflinger
Sonja
S
Research Institute of Molecular Pathology, Vienna Biocenter, Vienna, Austria.
Kesavan
Kamala
K
Fuxa
Martin
M
Hutter
Caroline
C
Heavey
Barry
B
Radtke
Freddy
F
Busslinger
Meinrad
M
D000818:Animals; D016175:B-Lymphocyte Subsets / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D002454:Cell Differentiation / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D019070:Cell Lineage / Q000235:genetics / Q000276:immunology; D002999:Clone Cells; D018920:Coculture Techniques; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology; D015536:Down-Regulation / Q000235:genetics / Q000276:immunology; D005786:Gene Expression Regulation / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation; D051757:PAX5 Transcription Factor; D051881:Receptor, Notch1; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism* / Q000502:physiology; D017154:Stromal Cells / Q000502:physiology; D016176:T-Lymphocyte Subsets / Q000166:cytology* / Q000378:metabolism / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology*
D004268:DNA-Binding Proteins; C497762:Notch1 protein, mouse; D051757:PAX5 Transcription Factor; C497282:Pax5 protein, mouse; D051881:Receptor, Notch1; D016693:Receptors, Antigen, T-Cell, alpha-beta; D016692:Receptors, Antigen, T-Cell, gamma-delta; D011956:Receptors, Cell Surface; D014157:Transcription Factors