Cdc42-mTOR Signaling Pathway Controls Hes5 and Pax6 Expression in Retinoic Acid-dependent Neural Differentiation

The conditional knockout of the small GTPase Cdc42 from neuroepithelial (NE) and radial glial (RG) cells in the mouse telencephalon has been shown to have a significant impact on brain development by causing these neural progenitor cells to detach from the apical/ventricular surface and to lose their cell identity. This has been attributed to the requirement for Cdc42 in establishing properapical/basal cell polarity and cell-cell adhesions. In the present study, we provide new insights into the role played by Cdc42 in the maintenance of neural progenitor cells, using the mouse embryonal carcinoma P19 cell line as a model system. We show that the ability of P19 cells to undergo the transition from an Oct3/4-positive, undifferentiated status to microtubule-associated protein 2-positive neurons and glial fibrillary acidic protein-positive astrocytes, upon treatment with retinoic acid (RA), requires RA-induced activation of Cdc42 during the neural cell lineage specification phase. Experiments using chemical inhibitors and RNA interference suggest that the actions of Cdc42 are mediated through signaling pathways that start with fibroblast growth factors and Delta/Notch proteins and lead to Cdc42-dependent mTOR activation, culminating in the up-regulation of Hes5 and Pax6, two transcription factors that are essential for the maintenance of NE and RG cells. The constitutively active Cdc42(F28L) mutant was sufficient to up-regulate Hes5 and Pax6 in P19 cells, even in the absence of RA treatment, ultimately promoting their transition to neural progenitor cells. The ectopic Cdc42 expression also significantly augmented the RA-dependent up-regulation of these transcription factors, resulting in P19 cells maintaining their neural progenitor status but being unable to undergo terminal differentiation. These findings shed new light on how Cdc42 influences neural progenitor cell fate by regulating gene expression.