RESUMO
Myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic Bcl-2 protein, regulates neural precursor cell (NPC) survival in both the developing and adult mammalian nervous system. It is unclear when during the neurogenic period Mcl-1 becomes necessary for NPC survival and whether Bax is the sole pro-apoptotic target of Mcl-1. To address these questions, we used the nervous system-specific Nestin-Cre Mcl-1 conditional knockout mouse line (Mcl-1 CKO) to assess the anti-apoptotic role of Mcl-1 in developmental neurogenesis. Loss of Mcl-1 resulted in a wave of apoptosis beginning in the brainstem and cervical spinal cord at embryonic day 9.5 (E9.5) and in the forebrain at E10.5. Apoptosis was first observed ventrally in each region and spread dorsally over time. Within the spinal cord, apoptosis also spread in a rostral to caudal direction following the path of differentiation. Breeding the Mcl-1 CKO mouse with the Bax null mouse rescued the majority of NPC from apoptosis except in the dorsomedial brainstem and ventral thoracic spinal cord where only 50% were rescued. This demonstrates that Mcl-1 promotes NPC survival primarily by inhibiting the activation of Bax, but that Bax is not the sole pro-apoptotic target of Mcl-1 during embryonic neurogenesis. Interestingly, although co-deletion of Bax rescued the majority of NPC apoptosis, it resulted in embryonic lethality at E13, whereas conditional deletion of both Mcl-1 and Bax rescued embryonic lethality. In summary, this study demonstrates the widespread dependency on Mcl-1 during nervous system development.
RESUMO
During neurogenesis, proliferating neural precursor cells (NPC) exit the cell cycle and differentiate into postmitotic neurons. The proteins that regulate cell survival through the stages of differentiation, however, are still poorly understood. Here, we examined the roles of the anti-apoptotic Bcl-2 proteins, Mcl-1 and Bcl-xL, in promoting survival as cells progress through the stages of neurogenesis in the mouse embryonic central nervous system. We used Nestin-mediated, nervous system-specific conditional deletion of mcl-1, bcl-x or both to identify their distinct and overlapping roles. Individual conditional deletion of mcl-1 (MKO) and bcl-x (BKO) suggested sequential roles in promoting cell survival during developmental neurogenesis. In the MKO embryo, apoptosis begins at embryonic day 10 (E10) in the proliferating NPC population throughout the entire developing nervous system. In the BKO embryo, apoptosis begins later at E11 within the postmitotic neuron populations. In the double (mcl-1 and bcl-x) conditional knockout (DKO), cell death extended throughout both proliferating and non-proliferating cell populations resulting in embryonic lethality at E12, earlier than in either the MKO or BKO. Apoptotic cell death of the entire central nervous system in the DKO demonstrates that both genes are necessary for cell survival during developmental neurogenesis. To determine whether Mcl-1 and Bcl-xL have overlapping anti-apoptotic roles during neurogenesis, we examined the impact of gene dosage. Loss of a single bcl-x allele in the MKO embryo exasperated apoptotic cell death within the NPC population revealing a novel anti-apoptotic role for Bcl-xL in proliferating NPCs. Cells were rescued from apoptosis in both the MKO and BKO embryos by breeding with the Bax null mouse line indicating that Mcl-1 and Bcl-xL have a common pro-apoptotic target during developmental neurogenesis. Taken together, these findings demonstrate that Mcl-1 and Bcl-xL are the two essential anti-apoptotic Bcl-2 proteins required for the survival of the developing mammalian nervous system.
