Effect of NeuroD2 expression on neuronal differentiation in mouse embryonic stem cells.

A basic helix-loop-helix transcriptional factor, NeuroD2, plays important roles in neuronal differentiation and survival. We introduced the tetracycline-dependent NeuroD2 expression system to embryonic stem (ES) cells and studied the role of NeuroD2 in the neuronal differentiation. The addition of doxycycline induced the expression of NeuroD2 after 24h and the differentiation to neurons after 3 days in ES cells, which are transfected with vectors composed of reverse tetracycline-controlled transactivator with cytomegarovirus promoter and NeuroD2 with tetracycline response element. Treatment with doxycycline for 3 days induced neuronal differentiation, but not within 1 day; furthermore NeuroD2 was detected in the nucleus 3 days after treatment, but also not within 1 day. The results suggest that the expression of NeuroD2 requires an appropriate period of about 3 days to elicit neuronal differentiation in ES cells.