Mcl-1 promotes neural precursor cell cycle exit and differentiation in the mouse embryonic brain

Abstract

Neural precursor cell (NPC) proliferation and apoptosis are key regulatory aspects of mammalian nervous system development. Although recent studies suggested these two processes to be interrelated, the molecular mechanisms behind this remain undefined. Here I show that myeloid cell leukemia-1 (Mcl-1), a Bcl-2 family member that is essential for the survival of NPCs also reduces NPC proliferation and promotes their terminal mitosis. I found that within 48 hours of in utero electroporating Mcl-1 in E13.5 mouse embryonic brains, the majority of NPCs transfected with Mcl-1 have migrated into the post mitotic conical plate, whereas control transfected NPCs are still within the pro liferating ventricular/subventricular zones. Analysis of proliferation by proliferating cell nuclear antigen (PCNA) immunohistochemistry revealed a 2-fold reduction in proliferating NPCs in the Mcl-1 treated brains. Immunohistochemistry for Tbrl, a marker for newborn neurons, showed a 50% increase in differentiated neurons in Mcl-1 treated brains. BrdU birthdating demonstrated that Mcl-1 overexpression results in a greater cohort of newborn neurons. Furthermore, Mcl-1 transfected NPCs gave rise to neurons in the deeper layers of the cortex than control transfected NPCs confirming an earlier birthdate. Similarly, transfection of Mcl-1 in NPCs in vitro promotes cell cycle exit. I showed that Mcl-1 interacts with key cell cycle regulators in NPCs, namely PCNA and Cdk1/Cyclin B1 complex. In addition, I found an increase in Cdk inhibitor p27Kip1 protein, a key promoter of cell cycle exit with Mcl-1 overexpression and a concomitant decrease in p27Kip1 in Mcl-1 conditional knockout NPCs, suggesting that Mcl-1 may modulate p27Kip1 protein to promote NPC differentiation. Finally I showed that p27Kip1 is required for Mcl-1 mediated NPC cell cycle exit, suggesting that Mcl-1 regulates NPC cell cycle through p27Kip1 activity. In summary, these results identify a novel function for Mcl-1 in promoting terminal mitosis of NPCs by influencing the cell cycle regulatory machinery.