Cell fate determination and differentiation during neurogenesis and myogenesis involve the sequential expression of several basic helix-loop-helix (bHLH) transcription factors. The expression of NeuroD1/2 and the expression of NSCL(Nh1h)1/2 are closely related in many developing peripheral and central neuronal cells, suggesting an epistatic relationship between these two bHLH transcription factor families during neurogenesis. To investigate this relationship, a murine neuroblastoma cell culture system and single/double knock-out (KO) mice of NeuroD1 and NeuroD2 were utilized for the gain-of-function and loss-of-function approaches, respectively. Both NeuroD1 and NeuroD2 were able to induce the transcription of NSCL1 in vitro: however, they were not able to activate NSCL2 transcription. The DNA-binding ability of NeuroD1 was essential for NSCL1 induction. To examine the epistatic relationship in vivo, we examined the expression of NSCL1 and NSCL2 in NeuroD1 and NeuroD2 KO mice and NeuroD1/2 compound MO mice by in situ hybridization, RT-PCR and Northern blotting. The expression of NSCL1 was lower in the NeuroD1 KO mice and was not further decreased in the double KO mice. However, the expression of NSCL2 did not change in either the single KO or double KO mice. These results demonstrate that NeuroD1 is an upstream regulator of the NSCL1 gene but not the NSCL2 gene in mice. In addition, NeuroD2 is not involved in this regulatory pathway in vivo. (C) 2012 Elsevier Inc. All rights reserved.