THE P53 FAMILY MEMBER, P63, REGULATES NEURAL PRECURSOR CELL SURVIVAL DURING CORTICAL DEVELOPMENT

Background: p63, a member of the p53 family of proteins, is involved in the regulation of naturally-occurring apoptosis in sympathetic neurons of the peripheral nervous system. Since data from our laboratory indicated that p63 is also expressed in stem cells and neurons within the developing brain, we hypothesized that p63 is involved in regulating the genesis and survival of developing neurons. Methods: As cortical neurogenesis is initiated at embryonic day 12, we knocked-down p63 levels in isolated murine cortical precursors byusing shRNA against p63 or by transfecting floxed-p63 precursors with Cre recombinase. We performed similar studies in vivo using in uteroelectroporation to express either p63 shRNA or Cre recombinase to acutely knockdown or genetically ablate p63. We then performed immunofluorescence for known markers of apoptosis, cell-division, and differentiation to assess the level of cell death, proliferation and neurogenesis. Results: Knock-down of p63 in vitro resulted in a 2-foldincrease in the death of precursors and neurons, associated with blunted neurogenesis but unaltered precursor proliferation. Coincident knock-down of p63 family members, p53, but not p73, rescued the elevated death suggesting that p63 and p53 antagonize each other to promote survival. Similar results were observed in vivo, where knockdown of p63 caused cell death and a decrease in the proportionof neurons in the cortical plate. Conclusions: These experiments indicate that p63 is required forthe survival of neural precursors and newly-born neurons, and for normal cortical development. Ongoing work will explore the environmental cues that regulate p63 during neurogenesis.