Id proteins facilitate self-renewal and proliferation of neural stem cells.

Members of helix-loop-helix (HLH) protein family of Id (inhibitor of differentiation) dimerize with bHLH transcription factors and function as negative regulators of differentiation during development. Most of inhibitory roles of Id proteins have been demonstrated in non-neural tissues, and their roles in the developing nervous system are not clearly demonstrated. In this study, we show that Id1, Id2, and Id3 increase self-renewing and proliferation potential of cortical neural stem cells (NSCs) while inhibiting neuronal differentiation. In electrophoretic mobility gel shift and luciferase assays, Id proteins interfered with binding of NeuroD/E47 complexes to the E-box sequences and inhibited E-box-mediated gene expression. Overexpression of Id proteins in NSCs increased both the number and the size of neurospheres in colony-forming assays. Expression of Hes1 and Hes5 was not increased by overexpression of Id proteins under the condition in which Nestin expression was increased. In utero electroporation of Id yielded higher numbers of Ki67-positive and Sox2-positive cells in the mouse embryonic brain. The study suggests Id proteins play independent roles in the maintenance of neural stem properties.