Acentriolar mitosis activates a p53-dependent apoptosis pathway in the mouse embryo

Centrosomes are the microtubule-organizing centers of animal cells that organize interphase microtubules and mitotic spindles. Centrioles are the microtubule-based structures that organize centrosomes, and a defined set of proteins, including spindle assembly defective-4 (SAS4) (CPAP/CENPJ), is required for centriole biogenesis. The biological functions of centrioles and centrosomes vary among animals, and the functions of mammalian centrosomes have not been genetically defined. Here we use a null mutation in mouse Sas4 to define the cellular and developmental functions of mammalian centrioles in vivo. Sas4-null embryos lack centrosomes but survive until midgestation. As expected, Sas4^−/− mutants lack primary cilia and therefore cannot respond to Hedgehog signals, but other developmental signaling pathways are normal in the mutants. Unlike mutants that lack cilia, Sas4^−/− embryos show widespread apoptosis associated with global elevated expression of p53. Cell death is rescued in Sas4^−/−p53^−/− double-mutant embryos, demonstrating that mammalian centrioles prevent activation of a p53-dependent apoptotic pathway. Expression of p53 is not activated by abnormalities in bipolar spindle organization, chromosome segregation, cell-cycle profile, or DNA damage response, which are normal in Sas4^−/− mutants. Instead, live imaging shows that the duration of prometaphase is prolonged in the mutants while two acentriolar spindle poles are assembled. Independent experiments show that prolonging spindle assembly is sufficient to trigger p53-dependent apoptosis. We conclude that a short delay in the prometaphase caused by the absence of centrioles activates a previously undescribed p53-dependent cell death pathway in the rapidly dividing cells of the mouse embryo.Centrioles are cylinders of triplet microtubules that provide the template for cilia and nucleate the centrosomes that act as microtubule organizing centers (MTOCs) at spindle poles and during interphase (1, 2). Genetic analysis has demonstrated that the biological roles of centrioles differ widely among organisms: Caenorhabditis elegans embryos without centrioles arrest at the two-cell stage, whereas zygotic removal of centrioles in Drosophila allows survival to adult stages (3–5). In humans, mutations in centriolar and centrosomal proteins are associated with microcephaly or microcephaly in the context of dwarfism (6–10). Abnormal numbers of centrioles are associated with cancer, although it is not clear whether abnormal centrosome number is a cause or an effect of tumorigenesis (1, 11–13). Studies in cultured cell lines have given conflicting results on the roles of vertebrate centrioles in mitosis, chromosome segregation, DNA damage response, and intercellular signaling (14–19), but the precise functions of mammalian centrioles have not been defined genetically.A small number of core proteins have been shown to be required for centriole biogenesis in organisms ranging from Chlamydomonas reinhardtii to human cells. Spindle assembly defective-4 (SAS4), one of these core proteins, acts at an early step in the assembly pathway, when it is required for the addition of tubulin subunits to the forming procentrioles; it also is required for recruitment of the pericentriolar material (PCM) to form the centrosome (3, 20, 21). Mutations in Sas4 block centriole formation in Drosophila and C. elegans, and mutations in human SAS4 (CPAP/CENPJ) cause Seckel syndrome (dwarfism with microcephaly) (3–6). siRNA knockdown of SAS4 in cultured mammalian cells was reported to cause formation of multipolar spindles (14).Here we use null mutations in Sas4 to define the cellular and developmental functions of centrioles in the mouse embryo. As expected, Sas4 is essential for formation of centrioles, centrosomes, and cilia and for cilia-dependent Hedgehog (Hh) signaling. Unexpectedly, Sas4^−/− embryos arrest at an earlier stage than mutants that lack cilia and show widespread cell death associated with strong up-regulation of p53 in most cells in the embryo. Genetic removal of p53 rescues both the cell death and the early lethality of Sas4^−/− mutants. Cell death in the mutants is not associated with defects in the cell-cycle profile, DNA damage response, or chromosome segregation. The data indicate that in Sas4^−/− mouse embryos prolonged prometaphase, caused by a delay in spindle pole assembly, triggers a previously uncharacterized checkpoint that activates p53-dependent apoptosis in vivo.