Background: The protein p63, a recently discovered member of the p53 family of proteins, is implicated in the maintenance and differentiation of stem cells in the epidermis and is involved in the regulation of naturally-occurring cell death in sympathetic neurons of the peripheral nervous system. Since initial data from our laboratory indicated that p63 is also widely expressed in stem cells and neurons within the developing brain, we assessed its involvement in regulating the genesis and survival of developing cortical neurons. As neurogenesis is initiated at embryonic day 12 (E12), we isolated cortical precursors from p63-/- embryos at E14 and cultured them for 2 days in vitro (DIV).
Methods: Based on immunocytochemistry to known markers of apoptosis and neurons, we assessed the level of cell death and neurogenesis.
Results: Compared to p63+/+ cortical precursors, p63-/- precursors from littermates showed a 50 % reduction in neuronal death, as assessed by the apoptosis marker, cleaved caspase 3. Interestingly, the proportion of neurons and astrocyte precursors, the latter identified by S100b was also reduced in p63-/- embryos, as compared to p63+/+ littermates.
Conclusions: These results suggest that p63 may be involved in the regulation of cell survival and in the differentiation of precursors into neurons and astrocytes. To assess the former, we overexpressed TAp63a, a full-length isoform of p63, in E12/13 cortical precursors and assessed the level of cell death after 2 DIV. Compared to control cells, cells transfected with TAp63a demonstrated a 2-fold increase in cell death. Ongoing work will characterize p63 involvement in differentiation of precursor cells into neurons and astrocytes. To assess if these findings are relevant in vivo, we will use p63flox,flox X Nextin-Cre mice, which have p63 specifically ablated in neural precursors. We will analyze the survival, proliferation, and fate of these p63-/- cells. Together, these studies will help to determine a role for p63 in neural proliferation and apoptosis, processes central to development and response to injury.