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Acentriolar mitosis activates a -dependent PNAS PLUS pathway in the mouse

Hisham Bazzi and Kathryn V. Anderson1

Developmental Program, Sloan–Kettering Institute, Memorial Sloan–Kettering Center, New York, NY10065

Edited by Brigid L. M. Hogan, Duke University Medical Center, Durham, NC, and approved March 11, 2014 (received for review January 10, 2014) are the -organizing centers of (dwarfism with microcephaly) (3–6). siRNA knockdown of SAS4 cells that organize and mitotic spindles. in cultured mammalian cells was reported to cause formation of are the microtubule-based structures that organize cen- (14). trosomes, and a defined set of , including spindle assembly Here we use null in Sas4 to define the cellular and defective-4 (SAS4) (CPAP/CENPJ), is required for biogenesis. developmental functions of centrioles in the mouse embryo. As The biological functions of centrioles and centrosomes vary among expected, Sas4 is essential for formation of centrioles, cen- , and the functions of mammalian centrosomes have not trosomes, and cilia and for cilia-dependent Hedgehog (Hh) sig- − − been genetically defined. Here we use a null in mouse naling. Unexpectedly, Sas4 / arrest at an earlier stage Sas4 to define the cellular and developmental functions of mamma- than mutants that lack cilia and show widespread death as- lian centrioles in vivo. Sas4-null embryos lack centrosomes but sur- −/− sociated with strong up-regulation of p53 in most cells in the vive until midgestation. As expected, Sas4 mutants lack primary embryo. Genetic removal of p53 rescues both the cell death and − − cilia and therefore cannot respond to Hedgehog signals, but other the early lethality of Sas4 / mutants. Cell death in the mutants developmental signaling pathways are normal in the mutants. is not associated with defects in the cell-cycle profile, DNA Sas4−/− Unlike mutants that lack cilia, embryos show widespread damage response, or segregation. The data indicate − − apoptosis associated with global elevated expression of p53. Cell that in Sas4 / mouse embryos prolonged , caused Sas4−/− p53−/− death is rescued in double-mutant embryos, dem- by a delay in spindle pole assembly, triggers a previously unchar- onstrating that mammalian centrioles prevent activation of a p53- acterized checkpoint that activates p53-dependent apoptosis BIOLOGY dependent apoptotic pathway. Expression of p53 is not activated in vivo. DEVELOPMENTAL by abnormalities in bipolar spindle organization, chromosome seg- regation, cell-cycle profile, or DNA damage response, which are Results normal in Sas4−/− mutants. Instead, live imaging shows that the Sas4-Null Embryos Arrest at Midgestation. Animals homozygous for duration of prometaphase is prolonged in the mutants while two a partial loss-of- mutation in Sas4/Cenpj/Cpap generated acentriolar spindle poles are assembled. Independent experiments by the International Knockout Mouse Consortium (IKMC) are show that prolonging spindle assembly is sufficient to trigger p53- viable but dwarfed and represent a model for human Seckel dependent apoptosis. We conclude that a short delay in the prom- syndrome (22). To define the precise functions of mammalian etaphase caused by the absence of centrioles activates a previously centrioles, we generated a null from the IKMC partial loss- undescribed p53-dependent cell death pathway in the rapidly divid- of-function allele (Materials and Methods). At embryonic day (E) ing cells of the mouse embryo. − − 8.5, Sas4 / embryos had a normal anterior–posterior body axis and specified all three germ layers (Fig. 1 A, C, and D and Fig. S1 − − entrioles are cylinders of triplet microtubules that provide A and B). All Sas4 / embryos arrested by E9.0, when they had Cthe template for cilia and nucleate the centrosomes that act made a heart and neural tube but failed to undergo embryonic as microtubule organizing centers (MTOCs) at spindle poles and during interphase (1, 2). Genetic analysis has demonstrated that Significance the biological roles of centrioles differ widely among : Caenorhabditis elegans embryos without centrioles arrest at the two-cell stage, whereas zygotic removal of centrioles in Dro- Centrioles form the core of centrosomes, which organize cilia sophila allows survival to adult stages (3–5). In humans, muta- and interphase and spindle microtubules in animal cells, but tions in centriolar and centrosomal proteins are associated with function has not been defined in in vivo. microcephaly or microcephaly in the context of dwarfism (6–10). We show that mouse embryos that lack centrioles and cen- Abnormal numbers of centrioles are associated with cancer, al- trosomes survive to midgestation, when they lack primary cilia though it is not clear whether abnormal centrosome number is and cilia-dependent signaling. Despite the absence of cen- a cause or an effect of tumorigenesis (1, 11–13). Studies in cul- trosomes, bipolar spindle formation, , cell-cycle profile, and DNA damage response are normal in the tured cell lines have given conflicting results on the roles of ver- mutants. Unlike mutants that lack cilia, most cells in acentriolar tebrate centrioles in mitosis, chromosome segregation, DNA embryos activate a p53-dependent apoptotic pathway. The damage response, and intercellular signaling (14–19), but the data show that mammalian centrioles promote the efficient precise functions of mammalian centrioles have not been defined and rapid assembly of the mitotic spindle and that a short genetically. delay in prometaphase activates a checkpoint that leads to A small number of core proteins have been shown to be re- p53-dependent cell death in vivo. quired for centriole biogenesis in organisms ranging from Chla-

mydomonas reinhardtii to human cells. Spindle assembly Author contributions: H.B. and K.V.A. designed research; H.B. performed research; H.B. con- defective-4 (SAS4), one of these core proteins, acts at an early tributed new reagents/analytic tools; H.B. and K.V.A. analyzed data; and H.B. and K.V.A. step in the assembly pathway, when it is required for the addition wrote the paper. of subunits to the forming procentrioles; it also is re- The authors declare no conflict of interest. quired for recruitment of the (PCM) to This article is a PNAS Direct Submission. form the centrosome (3, 20, 21). Mutations in Sas4 block 1To whom correspondence should be addressed. E-mail: [email protected]. centriole formation in Drosophila and C. elegans, and muta- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tions in human SAS4 (CPAP/CENPJ) cause Seckel syndrome 1073/pnas.1400568111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1400568111 PNAS Early Edition | 1of10 Downloaded by guest on October 2, 2021 − − Sas4 / Embryos Lack Centrioles and Cilia. The longer survival of − − Sas4 / embryos that lacked p53 facilitated the analysis of the cellular phenotypes caused by removal of SAS4. We used transmission electron microscopy (TEM) analysis of serial sec- − − − − tions of E8.5 Sas4 / p53 / neural to assess whether centrioles were eliminated in the mutants. We observed 90 centrioles in 134 sections of control mitotic and nonmitotic cells (Fig. 3 A and C and Fig. S3 A and C). In contrast, no centrioles − − − − were seen in 174 sections of Sas4 / p53 / embryos (Fig. 3 B and D and Fig. S3 B and D). Even in mutant mitotic cells (n = 4) in which condensed were in conformation, microtubules focused toward a pole that lacked centrioles in 23 serial sections (compare Fig. 3 B and D with Fig. 3 A and C). Centrioles are required for the formation of cilia in all eukar- yotes (25). The wild-type embryonic node has long cilia at E7.5 (Fig. S3E), but no cilia were detected by scanning electron mi- − − croscopy in the Sas4 / mutant node (Fig. S3F). Although cilia were present on most cells in wild-type embryos, no cilia were detected by immunostaining with the cilia markers ARL13B or − − acetylated α-tubulin (Ac-tub) in Sas4 / embryos at E8.5 −/− – Fig. 1. Sas4 mouse embryos arrest at mid-gestation. (A) Dorsal view of (Fig. 3 E H). wild-type and Sas4−/− embryos at E8.5. (B) Wild-type and Sas4−/− embryos at No focal organization of the PCM proteins γ-tubulin or peri- fl/- −/− E9.5. (C and D) Cross-sections of wild-type and -Cre; Sas4 embryos at centrin (PCNT) was detected in interphase cells of E8.5 Sas4 E8.5 stained for E-cadherin (red). Arrows indicate the neural plate (ecto- embryos (Fig. 3 E–H), confirming that removal of SAS4 dis- derm), arrowheads indicate the mesenchyme (mesoderm), and asterisks in- rupted the organization of the centrosome. Western blot analysis dicate the gut (endoderm). (Scale bars: 300 μm.)

− − turning (Fig. 1B). SAS4 was not detectable in E8.5 Sas4 / embryos (Fig. S1C). To test whether a maternal contribution of SAS4 allowed early development in the mutants, we generated maternal/zygotic Sas4-null mutants (Materials and Methods), which were indistinguishable from the zygotic-null embryos (Fig. S1D). Thus, mouse embryos can survive to midgestation and lay down the basic body plan in the complete absence of SAS4.

− − Widespread p53-Dependent Cell Death in Sas4−/− Embryos. Sas4 / embryos died more than a day earlier than mutants that lack cilia (23), indicating that centrioles in the developing embryo have functions in addition to acting as templates for cilia. DAPI- − − stained sections of Sas4 / embryos showed many pyknotic and fragmented nuclei. Levels of cleaved caspase-3 (Casp3) were elevated in the mutants, revealing that there was widespread apoptosis in all cell types of the mutant at E8.5 (Fig. 2 A and B). Because up-regulation of p53 is a well-known mediator of apo- ptosis, we examined p53 expression in mutant embryos. In con- + trast to wild-type embryos, in which p53-positive (p53 ) cells were rare, nuclear p53 was detected in nearly every healthy in- terphase cell in E8.5 mutant embryos (Fig. 2 D and E). By Western blot, the level of p53 was approximately fivefold higher − − in Sas4 / embryos than in wild-type embryos (Fig. 2F). − − To test whether the apoptosis in Sas4 / mutants was caused − − − − by elevated p53, we generated Sas4 / p53 / double-mutant embryos. The double mutants survived until at least E9.5, and + the number of Casp3 apoptotic cells was greatly reduced as − − − − compared with Sas4 / single mutants (Fig. 2 C and G). Sas4 / −/− p53 embryos at E9.5 had >20 somites, completed embryonic −/− – Fig. 2. Cell death and early lethality in Sas4 embryos are rescued by re- turning, and showed the randomized left right situs and the moval of p53. (A–C) Cleaved Casp3 (green) expression in cross-sections of −/− abnormal brain morphology characteristic of mutants that lack wild-type embryos (0.6 ± 0.4%, n = 3,073 from two embryos) (A), Sas4 −/− −/− cilia (Fig. 2G) (23). Ift88 mutants (24), which lack cilia but embryos (23 ± 4%, n = 3,150 from three embryos, P = 0.0048) (B), and Sas4 have normal centrosomes (Fig. S2 A and B), did not show ele- p53−/− embryos (2.7 ± 0.5%, n = 3,454 from two embryos, P = 0.0063) (C)at vated cell death or p53 expression (Fig. S2 D and E). Embryos E8.5. (D and E) p53 (red) is rare in wild-type embryos (0.4 ± 0.1%, n = 3,534 from two embryos) but is strongly expressed in Sas4−/− mutant embryos (91 ± homozygous for a mutation in another required for cen- n = P < F gt/gt 1%, 2,629 from three embryos, 0.0001). ( ) Western blot analysis of −/− −/− −/− triole biogenesis, centrosomal protein-152 (Cep152)(Cep152 ) p53 in E8.5 embryo extracts. (G)Wild-type,Sas4 , and Sas4 p53 em- (Fig. S2C), also showed increased apoptosis and p53 expression bryosatE9.5.P values are relative to wild type in each case. (Scale bars: (Fig. S2F). 30 μminA–E, 300 μminG.)

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−/− Fig. 3. Sas4 embryos lack centrioles, centrosomes, and cilia. (A–D) TEM of E8.5 neural folds. Microtubules (arrowheads) converge at metaphase spindle −/− −/− poles, where there are centrioles (arrow) in wild-type but not Sas4 p53 embryos. Asterisks mark the condensed chromosomes in A and B. C and D are magnifications of the boxed regions at the spindle poles in A and B, respectively. (E–H) Expression of the centrosomal and cilia markers in the mesenchymal cells in E8.5 wild-type and Sas4−/− embryos stained for γ-tubulin (γ-tub) or PCNT (green) and ARL13B (red) or Ac-tub (magenta). Cilia are absent, and focal of γ-tubulin and PCNT is detected only during mitosis in Sas4−/− mutants. (I–L) Expression of the PCM proteins CEP152 (red) and γ-tubulin (green) in −/− −/− −/− p53 MEFs [45 ± 5 arbitrary units (A.U.)] and Sas4 p53 MEFs (25 ± 9 A.U., P < 0.0001, n = 14 poles each). CEP152 is not detected in either interphase or −/− mitotic cells in Sas4 mutants. (Scale bars: 0.3 μminA–D,3μminE–L.)

− − showed that total γ-tubulin levels were comparable in E8.5 wild-type Hh signaling, Gli1 and Ptch1, were not expressed in Sas4 / and mutant embryos, indicating that γ- tubulin was still present mutants at E8.0–8.5 (Fig. S5 A and B) because of the absence of − − in the absence of a centriole (Fig. S4). Unlike Drosophila Sas4 / cilia. Dorsal–ventral patterning of the neural tube provides an mutants (5), both γ-tubulin and PCNT were enriched at the assay for different levels of Sonic hedgehog (Shh) signaling. The − − − − − − spindle poles of dividing cells in Sas4 / mutants at E8.5, albeit survival of Sas4 / p53 / mutants to E9.5 made it possible to at lower levels than in wild-type embryos (Fig. 3 E–H; see also analyze neural patterning in the absence of centrioles. The Fig. S8 A–F). Similarly, in mouse embryonic fibroblasts (MEFs) ventral neural cell types that depend on high or intermediate − − − − derived from Sas4 / p53 / embryos, γ-tubulin staining was not levels of Shh signaling (FOXA2 and NKX2.2) were absent in the detected during interphase but was present at lower levels at the double mutants (Fig. 4 A and B), and markers that normally are − − mitotic poles than in p53 / control cells (Fig. 3 K and L). In repressed by Shh (e.g., PAX6) spread into the ventral neural contrast, focal staining of CEP152, a PCM protein that is re- tube (Fig. 4 C and D). Thus, Shh-dependent neural patterning − − − − quired for centriole formation, was not detected in either in- was disrupted in Sas4 / p53 / mutants in the same way as in − − − − terphase or mitotic cells of Sas4 / p53 / MEFs (Fig. 3 I–L), mutants that lack cilia (23). indicating that CEP152 recruitment to the centrosome depends A number of components of Wnt signaling pathways are on SAS4. enriched at centrosomes, including β-catenin (19), AXIN1 (27), AXIN2 (28), and Dishevelled (29), and studies have suggested −/− Hh Signaling Is Disrupted in Sas4 Embryos, but Wnt Signaling Is that both canonical and noncanonical Wnt signaling depend on − − Normal. Primary cilia are required for responses to Hh family centrosomes or cilia (30–32). However, the phenotypes of Sas4 / ligands in the mouse (23, 26). As expected, two direct targets of embryos do not resemble those of Wnt pathway mutants. For

Bazzi and Anderson PNAS Early Edition | 3of10 Downloaded by guest on October 2, 2021 An objective assay for the activity of the noncanonical Wnt pathway is the polarized expression of planar cell polarity (PCP) proteins on the anterior and posterior faces of cells of the em- bryonic node (37–39), where noncanonical Wnt signaling is re- quired to polarize node cilia and initiate left–right asymmetry. − − Although Sas4 / mutants lack cilia and basal bodies, the PCP protein VANGL2 localized correctly to the anterior/posterior − − − − faces of Sas4 / p53 / node cells (Fig. 4 G–L and Fig. S5 E and F), indicating that the activity of the noncanonical Wnt pathway is normal in the absence of centrioles.

The Cell-Cycle Profile, Chromosome Segregation, and DNA Damage −/− Response Are Normal in Sas4 Embryos. To account for the up- regulation of p53 in the absence of centrioles, we evaluated functions that have been ascribed to centrioles and centrosomes that could regulate p53. Studies that used laser ablation, mi- crosurgery, or siRNA knockdown indicated that disruption of centrosomes in cultured mammalian cells causes a p38-, p53-, and -dependent G1 cell-cycle arrest (15, 16). Therefore we − − analyzed the by FACS of dissociated cells from Sas4 / embryos at E8.5. There were no obvious differences in cell-cycle − − profiles between Sas4 / and wild-type embryos (Fig. 5 A and B; G1: 27%, S: 60–63%, and G2/M: 8–12%); in particular, there was no G1/S arrest in the mutants. Consistent with the FACS results, Western blot analysis showed that the level of D1 − − (a G1 cyclin) was unchanged in Sas4 / embryos at E8.5 (Fig. 5C). Unlike the results in , active phospho-p38 (T180/ − − Y182) was not up-regulated in Sas4 / embryos at E8.5 (Fig. 5D). Many studies have correlated centrosomal aberrations with abnormal spindles and chromosomal instability (17, 40–42). − − − − Staining for α-tubulin in Sas4 / p53 / MEFs showed that bi- polar spindles formed in the absence of SAS4 (Fig. 5 E and F). Astral microtubules were present, although they were decreased in number in the double mutant MEFs (Fig. 5 E and F). To test whether the absence of centrioles caused , − − − − we examined chromosome number in cells from Sas4 / p53 / double-mutant embryos, reasoning that any aneuploid cells generated by removal of SAS4 would survive and accumulate in the absence of p53. We used - and 17-specific − − − − probes for FISH on dissociated cells from E8.5 Sas4 / p53 / − − double-mutant, wild-type, and p53 / embryos. Indistinguishable −/− levels of apparent aneuploidy were seen by this assay in wild-type Fig. 4. Hh, but not Wnt, signaling is disrupted in Sas4 mutants. (A–D) Hh- −/− −/− −/− −/− = dependent ventral cell types in the neural tube of E9.5 wild-type and Sas4 (6.0%), p53 (5.3%), and Sas4 p53 (6.7%) embryos (n −/− −/− −/− p53 embryos. In Sas4 p53 embryos floor plate cells (FOXA2, red) and 150 cells total from two embryos of each genotype) (Fig. 5 G and + V3 interneurons (NKX2.2, green) are absent (compare A and B), and PAX6 H). Supporting the conclusion that removal of SAS4 does not interneurons (green) occupy the ventral neural tube (compare C and D). (E cause detectable aneuploidy, metaphase chromosome counts − − and F) The activity of the canonical Wnt reporter TOPGAL (blue) is normal in were indistinguishable in cells from E8.5 Sas4 / and wild-type −/− E8.5 Sas4 mutants. (G–L) The PCP noncanonical Wnt signaling protein embryos (Fig. S6 A and B). VANGL2 (green) localizes to the anterior/posterior (horizontal) faces of the G–I Sas4−/− p53−/− A number of proteins in the DNA damage response pathway, cells in the embryonic node of both wild-type ( )and embryos including CHK1 and ATM, appear to be enriched at cen- (J–L) at E7.75. Rhodamine-conjugated phalloidin (red) marks F- at cell borders. (Scale bars: 300 μminA–F,3μminG–L.) trosomes (43), and it has been suggested that mutations in spe- cific centrosomal proteins disrupt the DNA damage response pathway (18). We did not detect any difference between E8.5 − − example, Wnt3 and Axin1 mutants arrest at gastrulation, and wild-type and Sas4 / embryos in the staining pattern of 53BP1, Wnt3a mutants are posteriorly truncated (33–35); neither phe- which marks the sites of DNA damage (Fig. 5 I and J). Western − − − − notype was seen in Sas4 / p53 / mutants (Fig. 2G), suggesting blot analysis showed that the level of another marker of DNA − − − − − − that canonical Wnt signaling was not strongly disrupted. Ex- damage, γ-H2AX, was not increased in Sas4 / or Sas4 / p53 / − − pression of TOPGAL, a transgenic reporter of canonical Wnt embryos (Fig. 5K). To test whether Sas4 / cells are able to ac- − − − − signaling, was indistinguishable in Sas4 / mutants and wild-type tivate the normal response to DNA damage, we treated Sas4 / − − − − embryos at E7.5 and E8.5 (Fig. 4 E and F and Fig. S5C). Ex- p53 / and p53 / MEFs with ionizing radiation for 1 h, which pression of Axin2, a direct target of canonical Wnt signaling, also increased the levels of γ-H2AX and pCHK1 (S345) to the same − − was normal in Sas4 / embryos at E7.5 (Fig. S5D). extent in mutants and controls (Fig. S6C). The cell cycle in both − − − − − − Mutations in the noncanonical Wnt pathway cause charac- Sas4 / p53 / and p53 / MEFs arrested in response to irradi- teristic morphological abnormalities; the most prominent defect ation, as assayed by decreased levels of phospho- H3 is the failure to close the entire neural tube posterior to the (pHH3) (Fig. S6C). Treatment with UV radiation or hydroxy- midbrain (craniorachischisis) (36). In contrast, the spinal neural urea for 2 h also induced a robust DNA damage response in − − − − − − − − tube in Sas4 / p53 / mutants was closed (Figs. 2G and 4 A–D). Sas4 / p53 / MEFs (Fig. S6D).

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−/− Fig. 5. Cell-cycle profile, chromosome segregation, and the DNA damage response are normal in Sas4 mutants. (A and B) Cell-cycle profiles of cells isolated −/− from E8.5 wild-type (A) and Sas4 (B) embryos. (C) Western blots show similar levels of in wild-type and mutant E8.5 embryo extracts. (D) Phospho-

−/− −/− −/− −/− BIOLOGY p38 (pp38, T180/Y182) is not elevated in E8.5 Sas4 embryo extracts. (E and F) Mitotic spindles from p53 (control) and Sas4 p53 MEFs stained with −/− −/− −/− α-tubulin (green) and PCNT (red). (G and H) Sample FISH signals from dissociated cells of p53 control and Sas4 p53 embryos at E8.5 with two copies of DEVELOPMENTAL −/− chromosome 12 (green) and two copies of (red). (I and J) Wild-type (I) and Sas4 (J) embryo sections at E8.5 (neural plate) stained with the DNA damage marker 53BP1. (K) Western blots of embryo extracts show that γ-H2AX is not elevated in Sas4−/− or Sas4−/− p53−/− cells at E8.5 relative to the respective wild types. For loading controls, α-tubulin was used for Sas4−/− and GAPDH for Sas4−/− p53−/−. (Scale bars: 3 μm.)

−/− Prometaphase Is Prolonged in Sas4 Embryonic Cells. Because of embryos) compared with wild type (5.4 ± 0.5%, n = 16,277 cells the localization of centrioles at mitotic spindle poles, we exam- from five embryos; P = 0.0014). Consistent with the increase in − − ined mitosis in the absence of centrioles in Sas4 / embryos. mitotic cell number, the level of (a mitotic cyclin) in- − − Embryo sections stained for the mitotic marker pHH3 (Fig. 6 A creased ∼1.6-fold in Sas4 / embryos (Fig. 6C). and B) showed a 70% increase in the fraction of mitotic cells in To follow the dynamics of mitosis in the embryo, we generated − − − − Sas4 / embryos at E8.5 (9.1 ± 0.8%, n = 19,212 cells from nine Sas4 / mutants that carried an H2B-GFP transgene (44) and

−/− + −/− Fig. 6. Prolonged mitosis in Sas4 embryos. (A and B) Cross-sections of E8.5 embryos show an increase in the number of mitotic cells (pHH3 ; red) in Sas4 −/− −/− −/− p53 embryos. (C) Western blots show an increase in cyclin B1 (arrow) in E8.5 Sas4 p53 embryos. Asterisks mark nonspecific bands. (D and E) Snapshots from time-lapse imaging of dividing cells in cultured E7.5 wild-type (D) and Sas4−/− (E) H2B-GFP+ embryos. (Scale bars: 30 μminA and B,3μminD and E.)

Bazzi and Anderson PNAS Early Edition | 5of10 Downloaded by guest on October 2, 2021 − − imaged the chromosomes of dividing cells in intact cultured mitosis in Sas4 / embryos (Fig. S8 E–H). The longer duration of embryos beginning at E7.5, a stage when mutant embryos prometaphase was confirmed by assay of the spindle assembly showed increased p53 and apoptosis (Fig. S7). In every case (n = checkpoint (SAC). The SAC proteins MAD2 and BUBR1 were 124 cells from five embryos), the condensed chromosomes suc- both enriched at in mutant cells during prom- cessfully segregated to two poles. We observed no examples of etaphase (Fig. S8 I–L). We conclude that during acentriolar multipolar spindles, and lagging chromosomes were observed in mitosis in mouse embryos prometaphase is extended because of only two cases in the mutant and in two cases in wild-type em- a slower alternative mechanism for spindle pole assembly. bryos (Fig. 6 D and E and Movies S1 and S2). Despite the successful completion of mitosis, live imaging Prolonged Mitosis Activates p53 and Apoptosis in the Wild-Type showed that the time from to was signifi- Embryo. It is remarkable that a short (∼10 min) increase in the − − cantly longer in Sas4 / than in wild-type embryos: Mitosis lasted duration of prometaphase appeared to cause induction of p53 in − − − − 30.1 ± 4.8 min in Sas4 / embryos (n = 52 cells from three Sas4 / embryos. In cultured cells, treatment with the micro- embryos) compared with 21.0 ± 2.9 min in wild type (P < 0.0001, tubule-depolymerizing agent nocodazole prolongs mitosis and n = 112 cells from three embryos) (Fig. 6 D and E). The delay activates p53 without affecting interphase progression (45, 46). occurred during prometaphase, when chromosomes organized in Therefore, to test whether a short delay in mitosis could activate rosette-like arrangements for ∼15 min in the mutant, compared p53 in the mouse embryo, we cultured E8.5 wild-type embryos with ∼6 min in wild type. In every mitosis, however, the mutant for 30 or 60 min in the presence of a low concentration (1 μM) of chromosome rosette resolved into two equal groups of chro- followed by a washout and then 2 or 4 h of culture (Table S1). After a 60-min treatment, 10.7% of the cells in mosomes that segregated to two poles. + treated embryos were p53 at 2 or 4 h after nocodazole removal, −/− + Spindle Pole Assembly in Sas4 Cells. The delay in anaphase onset but p53 cells were rare (1.5%) in control cultured embryos (Fig. − − + and chromosome segregation in Sas4 / embryos was paralleled 7 A and B and Table S1). The number of apoptotic Casp3 cells by delayed formation of two defined spindle poles, as assessed by was elevated at 4 h but not at 2 h after nocodazole removal (Fig. staining for the PCM proteins γ-tubulin, PCNT, and aurora ki- 7 C and D), suggesting that the cells delayed in mitosis up-reg- nase A (AURKA) (Fig. S8 A–H). Wild-type mitotic cells in the ulated p53 rapidly during the subsequent and initiated embryo invariably had two well-defined and brightly stained apoptosis shortly thereafter. A similar increase in nuclear p53 + γ-tubulin spindle poles throughout mitosis (Fig. S8A). In con- was obtained after 60-min treatment with 0.5 mM monastrol + trast, multiple small γ-tubulin foci or a faint cloud of γ-tubulin (Table S1), which inhibits the Eg5 and spindle assembly − − were observed at prometaphase in Sas4 / embryos (Fig. 3F and (46, 47). Incubation of wild-type embryos in 1 μM nocodazole for Fig. S8B). Despite the abnormal γ-tubulin foci during prom- only 30 min, which is the length of time that acentriolar cells etaphase, γ-tubulin always was focused at the two opposing poles spend in mitosis, led to elevated levels p53 in 6.1% of the cells at at anaphase (Fig. S8 C and D). PCNT and AURKA showed the 4 h after drug removal (Table S1). These data suggest that every same gradual coalescence at spindle poles as γ-tubulin during cell delayed in mitosis in this experiment activated a pathway

Fig. 7. Prolonged prometaphase activates p53 and apoptosis in the wild-type mouse embryo. (A–D) Cross-sections of cultured wild-type E8.5 embryos treated with vehicle (DMSO) (A and C)or1μM nocodazole for 1 h (B and D) and harvested 4 h after nocodazole removal. Approximately 11% of the cells in nocodazole-treated embryos are p53+ (red) (B). The number of Casp3+ (green) cells also was elevated in nocodazole-treated embryos (D), indicating increased apoptotic cell death. (E) Model for the activation of p53 and apoptosis by acentriolar mitosis. (Upper) Mitotic cells in wild-type embryos use centrioles to recruit more PCM and form mature centrosomes that organize microtubules and establish the bipolar mitotic spindle during prometaphase. The chromo- somes are aligned quickly and efficiently at the metaphase plate of a well-structured bipolar spindle. Mitosis proceeds normally, and p53 and Casp3 are not −/− up-regulated in the daughter cells. (Lower) Mitotic cells in Sas4 embryos lack centrioles and rely primarily on a -based pathway to organize the mitotic spindle. The establishment of bipolar spindle poles is slower in the absence of centrioles, but acentriolar cells eventually assemble a bipolar spindle and align the chromosomes at the metaphase plate, followed by proper chromosome segregation. However, the daughter cells of the acentriolar division retain the memory of the prolonged prometaphase and up-regulate p53, which increases the probability of cell death (marked by Casp3 expression). (Scale bars: 100 μm.)

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1400568111 Bazzi and Anderson Downloaded by guest on October 2, 2021 that led to elevated levels of p53. We conclude that interference Although the prominent location of centrosomes at the spin- PNAS PLUS with mitotic spindle assembly in the mouse embryo by preventing dle poles led to the belief that they are required for accurate microtubule polymerization, by inhibiting Eg5, or by removing chromosome segregation, experiments have shown that either centrioles causes a prolonged mitosis that leads to p53-dependent centrosomes or chromosomes can act as the prime organizers of apoptosis. the spindle pole (59). The early mitoses in the preimplantation mouse embryo are acentriolar, demonstrating that the mouse has Discussion robust machinery to support acentriolar mitosis (60–62). The Sas4-null mutant mouse embryos lack centrioles and die at onset of gastrulation correlates with more rapid proliferation, midgestation with disrupted Hh signaling, elevated levels of p53, with average cell-cycle times of 4–8 h and cell cycles as fast as 2 h and increased apoptotic cell death. Mutations in three other in regions of the epiblast (63, 64). In our hands, the highest − − mouse that now are known to be required for centriole in Sas4 / E7.5 embryo is in the epiblast, whereas duplication, Plk4/Sak, Sil/Stil, and Rotatin/Ana-3, were shown – the mitotic index is lowest in the endoderm (Table S2). The previously to cause arrest at midgestation (48 52). Defects in difference in proliferation rate between the germ layers parallels Hh-dependent patterning were described in Sil/Stil mutant em- the higher expression of p53 and Casp3 in the epiblast than in bryos (53), and increased cell death was observed in Plk4 and the endoderm (Fig. S7B). These data suggest that acentriolar Rotatin mutants (48, 50). We also observed midgestation lethality mitosis is most likely to cause p53-dependent cell death in rap- associated with increased cell death and up-regulation of p53 in idly proliferating cell types. mutant embryos homozygous for a gene trap allele of Cep152, − − Live imaging showed that Sas4 / cells take about 50% longer which encodes another core protein of the centriole duplication to complete mitosis than wild-type cells because of an increase pathway. Thus, the absence of centrioles, rather than the absence ∼ ∼ of SAS4 per se, triggers up-regulation of p53, which causes in the length of prometaphase from 6to 15 min. During the widespread cell death and leads to midgestation lethality in the prolonged prometaphase, the chromosomes are arranged in a mouse embryo. rosette that could easily be mistaken for a monopolar spindle. The centrosome has been thought of as a signaling platform However, the live imaging shows this phase is transient and organized by the centriole (54). Many developmental pathways resolves to give two distinct spindle poles, paralleling the gradual are required for survival to midgestation, and some of these have assembly of two distinct PCM foci at the poles. The coalescence been inferred to be dependent on cilia or centrosomes. Never- of PCM foci appears to be similar to the gradual fusion of BIOLOGY

theless, we observe that specification of the anterior–posterior MTOCs during the acentriolar cell cycles in the preimplantation DEVELOPMENTAL body axis, which requires Wnt, Nodal, BMP, and FGF signaling, mouse embryo (61). A delay in mitosis also was reported in is normal in the absence of centrioles and centrosomes. It has Drosophila Sas4 mutants: Mitosis takes 30–40% longer in Dro- been suggested that centrosomes are important in directed cell sophila Sas4 mutant than in wild-type cells because migration (55), but the polarized collective migration of the of a prolonged phase of spindle assembly (5), although cell death anterior visceral endoderm (56) is normal in the absence of was not reported in Drosophila Sas4 mutants. centrioles, as shown by the formation of a single anterior–pos- The role of centrioles in microtubule organization appears to − − terior body axis in Sas4 / embryos. Similarly, the apical–basal be remarkably conserved in evolution. Centrioles are required oscillations of nuclei in pseudostratified epithelia (interkinetic for the formation of cilia in every studied (25). Unlike nuclear migration) are thought to depend on an apically an- results obtained in cell-culture studies using vertebrate cells, chored centrosome (57), but restriction of mitotic cells to the mouse centrioles do not appear to be essential for bipolar spindle −/− apical surface of the neural epithelium is normal in Sas4 formation or proper chromosome segregation (14, 17). Similar to mutants (Fig. 6 A and B). Drosophila zygotic mutants (5), the mouse embryo can proceed −/− The majority of cells in E8.5 Sas4 embryos express high through many cell divisions without centrioles, but in the mouse levels of p53. Increased p53 can be caused by DNA damage, but − − the delay in mitosis leads to an efficient up-regulation of p53 that there is no detectable increase in DNA damage in Sas4 / em- −/− does not occur in Drosophila. bryos, and Sas4 mutant cells can activate the DNA damage It is remarkable that a delay in prometaphase of only about 10 response pathways normally. Aneuploidy also can activate p53, min in acentriolar cells in the mouse embryo was sufficient to – −/− but live imaging of E7.5 8.5 Sas4 embryos showed that bi- increase the level of p53 by approximately fivefold (Figs. 2F and polar spindles formed and that chromosomes segregated suc- 6E). A short treatment of wild-type embryos with nocodazole cessfully in every case observed. FISH analysis confirmed that also caused rapid up-regulation of p53 followed by cell death. Sas4 the rate of aneuploidy is not elevated in mutants. Our Thus, the data indicate that a short delay in mitosis, caused results contrast with experiments that indicated that centrosome by either absence of centrioles or disruption of mitotic micro- amplification in the mouse central nervous system leads to the tubules, initiates a p53-dependent apoptotic pathway in the em- formation of multipolar spindles, aneuploidy, cell death, and bryo (Fig. 7E). Interference with microtubule dynamics during microcephaly (42). Removal of p53 in the background of mul- tiple centrosomes led to only a partial rescue (∼12%) of brain mitosis of cultured human cell lines also causes increased levels size (42). Thus, the absence of centrosomes and centrosome of p53 in the absence of DNA damage but leads to a p38/p21- amplification appear to have very different consequences. dependent G1 cell-cycle arrest rather than apoptosis; this cell- Because p53 is expressed at high levels in the majority of cells cycle arrest is seen only after treatments that caused arrest in > in Sas4-null mutants, but DNA damage and aneuploidy were not prometaphase for 1.5 h (46). We suggest that a prolonged mi- detected, we conclude that some other defect caused by the tosis initiates a common pathway that up-regulates p53 after exit absence of centrioles is responsible for the up-regulation of p53 from mitosis; the outcome of p53 up-regulation in cultured cells is and subsequent cell death. Our data do not rule out the possi- cell-cycle arrest, whereas the outcome in the embryo is apoptosis. bility of a low frequency of DNA damage or chromosome loss, The precise molecular mechanism that activates p53 could be the which have been observed in the absence of centrosomes in other prolonged activation of the SAC, mislocalization of a PCM com- species (58) and in Sas4 partial loss-of-function mutants (22). ponent, or inappropriate activity of a cell-cycle regulator; eluci- Indeed, we assume that the function of p53 pathway activation is dation of this mechanism will be important for understanding to prevent the survival of cells in the embryo that have the po- this previously unidentified role of the p53 tumor suppressor in tential to experience genomic aberrations. maintenance of genomic integrity.

Bazzi and Anderson PNAS Early Edition | 7of10 Downloaded by guest on October 2, 2021 Materials and Methods . The following antibodies were used in this study, The rabbit anti- Animals and Genotyping. The Sas4 knockout-first ES cell line (EPD0028_7_G05) SAS4 was a gift from Pierre Gönczy (Institut Suisse de Recherches tm1a(EUCOMM)Wtsi was obtained from the IKMC [Cenpj ](www.mousephenotype. Experimentales sur le Cancer, Lausanne, Switzerland) (1/1,000 for Western org/martsearch_ikmc_project/martsearch/ikmc_project/34777). The null allele blots). The rabbit anti-ARL13B (1/2,000) was described previously (76). The tm1a(EUCOMM)Wtsi rabbit anti-VANGL2 antibody was a gift from Mireille Montcouquiol (Uni- was generated by crossing Cenpj mice to CAGG-Cre mice (65). versity of Bordeaux, Bordeaux, France) (1/1,000) (77). The following anti- Actin-FLP transgenic mice [B6.Cg-Tg(ACTFLPe)9205Dym/J; Jax stock no. 005703] +/fl bodies were from Sigma-Aldrich: mouse anti–γ-tubulin (clone GTU-88, were used to excise the gene trap and obtain the Sas4 (fl, floxed allele) 1/1,000 for immunofluorescence, 1/5,000 for Western blots), mouse anti– conditional allele. Sox2-Cre transgenic mice were used to delete Sas4 from all α-tubulin (1/1,000 for immunofluorescence, 1/10,000 for Western blots), embryonic tissues excluding the extraembryonic lineages (66). To generate + +/− mouse anti–acetylated α-tubulin (1/1,000), and rabbit anti-CEP152 (1/200). maternal/zygotic Sas4 mutants, ZP3-Cre/ (67) Sas4 males were crossed to fl/fl fl/- + The following antibodies were from Developmental Studies Hybridoma Sas4 females to generate Sas4 ZP3-Cre/ females, which were crossed to +/− −/− Bank: mouse anti-PAX6 (1/10) and mouse anti-NKX2.2 (1/10). The following Sas4 males to give 50% maternal/zygotic Sas4 mutant embryos. The tm1a(EUCOMM)Wtsi antibodies were from Santa Cruz Biotechnology: mouse anti-cyclin D1 (A12, Cep152 knockout-first ES cell line [EPD0061_1_E05, Cep152 ]was 1/200), mouse anti-cyclin B1 (1/500), and rabbit anti–N-cadherin (1/200). The obtained from IKMC (www.mousephenotype.org/martsearch_ikmc_project/ following antibodies were from Cell Signaling: mouse anti-p53 (1/200 for martsearch/ikmc_project/30116). H2B-GFP transgenic mice were a gift from immunofluorescence), rabbit anti–γ-H2AX (1/1,000), rabbit anti-pCHK1 (S345; Anna-Katerina Hadjantonakis (Sloan–Kettering Institute, New York) (44). tm1Tyj 1/1,000), rabbit anti-pCHK1 (S317; 1/1,000), mouse anti-AURKA (1/200), and Mice carrying the Trp53 null allele (Jax stock no. 002080) were a gift ftm1Brn rabbit anti-pp38 (T180/Y182) and anti-p38 (1/1,000). The following antibodies from Andrea Ventura (Sloan–Kettering Institute, New York). Trp53 were from Abcam: rabbit anti–γ-H2AX (1/1,000) and rabbit anti-FOXA2 (1/ mice (Jax stock no. 008462) were a gift from David Solit (Sloan–Kettering 1,000). The following antibodies were from BD Biosciences: mouse anti-MAD2 Institute, New York). The TOPGAL Wnt-reporter and the Ptch-LacZ knockin fl/fl (1/200) and mouse anti-BUBR1 (1/200). The following antibodies were from mice were previously described (68, 69). Conditional Ift88 mice were a gift Millipore: rabbit anti–phospho- (1/500 for immunofluorescence, 1/ of Bradley Yoder (University of Alabama at Birmingham, Birmingham, AL) 2,000 for Western blot). Rabbit anti-pericentrin (1/5,000) was from Covance. (24). Phenotypes were analyzed in the FVB/NJ background. Genotyping was Other antibodies used were rabbit anti–cleaved caspase-3 (1/200, Promega), carried out using standard PCR protocols. Animal experiments were approved rabbit anti-p53 (CM5; 1/500 for Western blot and 1/200 for immunofluores- by the institutional animal care and use committee at the Sloan–Kettering cence; Leica Microsystems), and rabbit anti-53BP1 (1/1,000, Novus Biologicals). Institute. Secondary antibodies for immunofluorescence were Alexa Fluor 488-, 568-, 594-, 633-, or 647-conjugated ( Technologies) and used at 1/1,000. Immunofluorescence and Embryo Culture and Imaging. Cryosections of em- Secondary antibodies for Western blot were from GE Healthcare and – μ bryos were prepared using standard conditions (70). Cryosections (8 12 m) used at 1/10,000. Rhodamine-conjugated phalloidin (1/400) was obtained − were postfixed in methanol at 20 °C for 20 min for centrosomal staining or from Invitrogen. were used directly for immunofluorescence. MEFs were fixed in methanol for centrosomal staining or in 4% (wt/vol) paraformaldehyde (PFA) for 10 Scanning Electron Microscopy and TEM. Embryos for scanning electron mi- min for nuclear staining followed by methanol. For α-tubulin staining, the croscopy and TEM were dissected in 1× PBS at room temperature and directly × cells were fixed in 4% PFA in 1 PBS at 37 °C for 5 min. Images were fixed in half-strength Karnovsky’s fixative (2% PFA, 2.5% glutaraldehyde, obtained using a Zeiss Axioplan2 wide-field microscope equipped with an 0.1 M cacodylate buffer) (Electron Microscopy Sciences) overnight or longer MRC Axiocam camera (Zeiss) or a Leica SP2 or SP5 confocal microscope (Leica at 4 °C and processed as previously described (23, 78). For TEM, embryos Microsystems). Confocal images were analyzed using the Volocity software were postfixed in tannic acid to enhance microtubule visibility (79). TEM package (Improvision). Nuclei were counted and intensity was sections were observed using a JEOL 1200EX transmission electron micro- measured using ImageJ (National Institutes of Health). scope at 80 kV. For scanning electron microscopy, embryos were observed – For embryo cultures, embryos were dissected at E7.5 8.5, with their yolk using Zeiss SUPRA 25 FESEM. sac and ectoplacental cone intact, at 37 °C with DMEM:F12 supplemented with 10% (vol/vol) FBS and 1% penicillin-streptomycin. For the nocodazole Cell-Cycle Analysis and DNA Damage Studies. Embryos were dissected in cold μ experiments, embryos at E8.5 were cultured in 1 M nocodazole or 0.5 mM 1× PBS and dissociated in 0.25% trypsin/EDTA (Invitrogen) for 20–30 min at monastrol (Sigma-Aldrich) in rat serum for 30 or 60 min, washed, and then 37 °C with occasional trituration with a fine pipette. The dissociated cells incubated in rat serum for an additional 2 or 4 h. were washed in 1× PBS and fixed in 70% ethanol at −20 °C until the time of + For live imaging, H2B-GFP embryos at E7.75 were inserted into a hole analysis. Propidium iodide staining was used for DNA content analysis on created in a freshly prepared collagen matrix (BD Biosciences) on a glass- a FACSCalibur machine (BD Biosciences). FlowJo software was used to ana- bottomed, 35-mm dish and were covered with rat serum. The embryos were lyze the cell-cycle data. Both the Watson Pragmatic model and Dean–Jett– imaged on an inverted Leica SP5 confocal microscope equipped with an Fox models were used and showed similar distributions of cell-cycle phases. – incubation chamber at 37 °C and CO2 for 6 12 h with frames taken every 3 or The experiment was performed twice on pooled embryos. In the experiment −/− 5 min. The resulting time-lapse movies were analyzed using the Volocity shown, n = 9 wild-type embryos and n = 16 Sas4 mutant embryos. − − −/− −/− (Improvision) or Imaris software (Bitplane). Control p53 / MEFs and Sas4 p53 MEFs (passage 4–5) were irradi- MEFs were prepared from E9.5 embryos by removing the heart and trit- ated with a dose of 10 Gy using an X-Rad 225 machine (Precision X-Ray) and urating before seeding onto cell culture-treated 12-well plates. MEFs were were harvested 1 h after irradiation. Higher-passage MEFs (passage 20) were cultured in DMEM medium (Invitrogen) containing 10% FBS and 1% penicillin- subjected to UV irradiation using a StrataLinker 2400 machine (Agilent streptomycin. Technologies) at 20 j/m2 or hydroxyurea (Sigma-Aldrich) treatment at 5 mM and were harvested 2 h after irradiation or treatment. LacZ Staining, in Situ Hybridization, and FISH. β-Galactosidase activity was detected using standard described protocols (71). The Brachyury, Gli1, and Chromosome Spreads. Embryos were dissected for culture at E8.5 as described Axin2 in situ probes were previously described (72–74). Whole-mount in situ above and were incubated in embryo culture medium supplemented with hybridization was performed on embryos following standard methods (70). 10 μM colcemid (Roche Applied Science) for 4 h to enrich for mitotic meta- The embryos were photographed using an HRC Axiocam (Zeiss) fitted onto phases. Six to seven mutant or three wild-type embryos were genotyped, a Leica stereomicroscope (Leica). FISH was performed on dissociated em- pooled, and incubated in 0.25% trypsin/EDTA (Invitrogen) for ∼20–30 min at bryos at E8.5 (see Cell-Cycle Analysis below for details) using probes corre- 37 °C for dissociation. After the trypsin was inactivated in culture medium sponding to BAC clones mapping to chromosomes 12 and 17 according to and the cells were washed, the dissociated cells were incubated at 37 °C in published methods (75). 0.1 M KCl hypotonic solution for ∼25 min. The cells then were fixed in 3:1 methanol:acetic acid mixture on ice and spread on slides for metaphase Western Blot Analysis. Embryos were dissected in cold 1× PBS with 0.1% preparations according to described protocols (80). The slides were dried Tween-20 and were frozen at −80 °C to be lysed later in Laemmli buffer overnight and stained with DAPI, and the chromosomes were imaged using (BioRad). Western blots were performed according to standard protocols. a63× oil objective on a Leica SP5 confocal microscope (Leica). The luminescent detection reaction was performed using ECL Plus or Prime reagent (Amersham) according to the manufacturer’s recommendations. Statistical Analysis. Two groups or more of data were compared using a two- Relative band intensities was quantified using ImageJ (National Institutes tailed Student t test or ANOVA with multiple comparisons (Dunnett’s test) of Health). with a cutoff for significance of <0.05 (Prism software, GraphPad). The data

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1400568111 Bazzi and Anderson Downloaded by guest on October 2, 2021 are presented as the mean ± SD in all cases except in Fig. S5 E and F where images; Prasad Jallepali, Andrew Koff, Bryan Tsou, John Petrini, and their labo- PNAS PLUS the error bars represent SEM. The detailed analyses of the intensity of the ratories for reagents and advice; Anna-Katerina Hadjantonakis for the H2B-GFP −/− signals of VANGL2 and F-actin using ImageJ was described previously (39). mice; Elizabeth Lacy for Actin-FLP mice; Andrea Ventura for p53 mice; David fl/fl Solit for p53 mice; and Bradley Yoder for Ift88 mice. We thank Bryan Tsou, ACKNOWLEDGMENTS. We thank the Mouse , , Molec- Prasad Jallepalli, Tim Bestor and members of the K.V.A. laboratory for comments ular Cytology, and Electron Microscopy core facilities at Memorial Sloan– on the manuscript. This work was supported by National Institutes of Health Kettering Cancer Center. We thank Kunihiro Uryu of the electron micros- Grant NS044385 (to K.V.A.) and Memorial Sloan–Kettering Cancer Center Sup- copy core facility at Rockefeller University for help with TEM; Pierre Gönczy port Grant P30 CA008748. H.B. was supported by National Kidney Foundation for the SAS4 antibody; James Mahaffey for analysis of planar polarity and National Research Service Award postdoctoral fellowships.

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