Control of adult neural stem cell properties (aNSCs) by the retinoblastoma protein, pRb -

Abstract

Neurogenesis is an ongoing developmental process throughout life in the adult mammalian brain; however, adult Neural Stem Cells (aNSCs)-progenitors display a low proliferative potential and restricted differentiation capacity in vivo. During development, the tumor suppressor gene, pRb, regulates distinct aspects of neurogenesis including neuronal proliferation, differentiation and migration. We have recently investigated the role of Rb in adult neurogenesis and found that it regulates the rate of proliferation of aNSCs-progenitors that are found in the adult subventricular zone (aSVZ) without affecting subsequent neuronal differentiation in the olfactory bulb. Here, we examined how Rb controls the properties of aNSCs-progenitors in culture including their proliferation rate, self-renewal capacity and differentiation potential. To do this, we induced a temporal deletion of Rb specifically in aNSCs-progenitors in 8 week-old mice using a Nestin-CreERT2-YFP Tamoxifen-inducible system and Rbfloxed-floxed mice. 5 days following treatment, we dissected and dissociated the aSVZ tissue and performed neurosphere assays from Rb+-- (control) and Rb--- (mutant) animals. Cells were cultured at low and high densities in media supplemented with FGF2 and EGF for 6 days before passaging, and, cell sorting was performed toisolate recombined or green fluorescent cells only. In addition, we conducted birth-dating experimentsin vivo using Bromodeoxyuridine (BrdU) to study the self-renewal property of aNSCs in the absence of Rb. Our results showed that:1)Rb controls the proliferation of adult progenitors as evidenced by the significant increase in the total number and the average size of primary neurospheres generated from Rb--- NSCs versus Rb+-- NSCs. This difference was even more pronounced among recombined cells isolated by cell sorting.2)Rb does not seem to regulate the self-renewal capacity of aNSCsince the number of secondary neurospheres generated from these cells in culture did not change in the absence of Rb. This was further