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

ABSTRACT

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 alpha beta vs. gamma delta 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 (alpha CD3)/(alpha CD28) 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.