Pitx2 expression induces cell cycle exit and p21 expression in neural stem cells

Cortical development is a complex process that involves many events including proliferation, cell cycle exit, and differentiation that need to be appropriately synchronized.. Neural stem cells (NSCs) isolated from embryonic cortex are characterized by their ability of self-renewal under continued maintenance of multipotency. The G1 phase of the cell cycle is mostly associated with cell cycle arrest and cell differentiation. Cell cycle progression and exit during development is regulated by numerous factors, including cyclins, cyclin dependent kinases and their inhibitors. In this study, we exogenously expressed the homeodomain transcription factor Pitx2, usually expressed in postmitotic neurons of the embryonic cortex, in NSCs with low expression of endogenous Pitx2, and found that Pitx2 expression induced a rapid decrease in proliferation associated with an accumulation of NSCs in G1 phase. A search for potential cell cycle inhibitors responsible for such cell cycle exit of NSCs revealed that Pitx2 expression caused a rapid and dramatic (≈20-fold) increase in expression of the cell cycle inhibitor p21Cip. In addition, Pitx2 bound directly to the p21Cip promoter as assessed by chromatin immunoprecipitation (ChIP) in NSCs. Surprisingly, Pitx2 expression was not associated with an increase in differentiation markers, but instead the expression of nestin, associated with undifferentiated NSCs, was maintained. Our results suggest that Pitx2 directly regulates p21Cip expression and induces cell cycle exit in neural progenitors.