Characterization of the transcriptional expression of Notch-1 signaling pathway members, Deltex and HES-1, in developing mouse thymocytes

The Notch transmembrane protein is involved in a broad range of different developmental pathways in vertebrates and invertebrates. Targeted thymocyte expression of the Notch-1 intracellular domain has been shown to affect lineage commitment decisions such as those involving T cell vs. B cell, thymocyte alphabeta vs. gammadelta TCR, as well as CD4 vs. CD8 thymocyte commitment. In this paper. we quantitatively characterize thymocyte RNA expression of two purported transcriptional markers of Notch-1 signaling activity, Deltex and HES-1. Using a semiquantitative RTPCR approach, we show that both Deltex and HES-1 transcriptional levels are developmentally regulated as thymocytes mature from the earliest CD4/CD8 double negative thymocyte stage, through the intermediate CD4/CD8 double positive stage, and finally to the mature CD4 or CD8 single positive stage, Deltex and HES-1, despite both being transcriptional markers of Notch-1 activity, express different patterns of transcriptional activity among the thymocyte subsets. Neither treatment with combined alphaCD3/alphaCD28 antibodies nor the combination of the phorbol ester PMA and calcium ionophore ionomycin affects expression of Deltex in immature thymocytes however, PMA/ionomycin treatment does downregulate expression of HES-1, an affect mostly mediated by ionomycin. Finally, a difference in HES-1 expression is seen between CD4/CD8 double positive thymocytes isolated from wild-type vs. MHC class I/II deficient mice, suggesting that Notch-1 activity is modulated during, in vivo TCR/MHC-ligand selection events. (C) 2002 Published by Elsevier Science Ltd.