Delta-like 4-mediated Notch signaling is required for early T-cell development in a three-dimensional thymic structure

Delta-like 4 (D114)-mediated Notch signaling is critical for specifying T-cell fate, but how D114-mediated Notch signaling actually contributes to T-cell development in the thymus remains unclear. To explore this mechanism in the thymic three-dimensional structure, we performed fetal thymus organ culture using D114-deficient mice. DN1a/b-FDN2mt cells, which had not yet committed to either the alpha beta T or gamma delta T/NK cell lineage, did not differentiate into the alpha beta T-cell lineage in D114-deficient thymus despite the lack of cell fate conversion into other lineages. However, DN3 cells efficiently differentiated into a later developmental stage of alpha beta T cells, the double-positive (DP) stage, although the proliferation was significantly impaired during the differentiation process. These findings suggest that the requirement for Notch signaling differs between the earliest and pre-TCR-bearing precursors and that continued Notch signaling is required for proper differentiation with active proliferation of alpha beta T lineage cells. Furthermore, we showed that Notch signaling increased the c-Myc expression in DN3 cells in the thymus and that its overexpression rescued the proliferation and differentiation of DN3 cells in the D114-null thymus. Therefore, c-Myc plays a central role in the transition from stage DN3 to DP as a downstream target of Notch signaling.