Gradual centriole maturation associates with the mitotic surveillance pathway in mouse development

Abstract

Centrosomes, composed of two centrioles and pericentriolar material, organize mitotic spindles during cell division and template cilia during interphase. The first few divisions during mouse development occur without centrioles, which form around embryonic day (E) 3. However, disruption of centriole biogenesis in Sas-4 null mice leads to embryonic arrest around E9. Centriole loss in Sas-4^-/- embryos causes prolonged mitosis and p53-dependent cell death. Studies in vitro discovered a similar USP28-, 53BP1-, and p53-dependent mitotic surveillance pathway that leads to cell cycle arrest. In this study, we show that an analogous pathway is conserved in vivo where 53BP1 and USP28 are upstream of p53 in Sas-4^-/- embryos. The data indicate that the pathway is established around E7 of development, four days after the centrioles appear. Our data suggest that the newly formed centrioles gradually mature to participate in mitosis and cilia formation around the beginning of gastrulation, coinciding with the activation of mitotic surveillance pathway upon centriole loss.

Keywords: 53BP1; SAS-4; USP28; centrosomes; p53.

Figure 1. The mitotic surveillance pathway is conserved in the mouse in vivo

1. A

   Gross morphology of control (Ctrl), Sas‐4 ^−/−, Sas‐4 ^−/− 53bp1 ^−/−, and Sas‐4 ^−/− Usp28 ^−/− embryos at E9.5. At least five embryos were considered per genotype and all the double mutant embryos exhibited the rescue criteria mentioned in the text. Scale bar = 500 μm.

2. B, C

   Immunostaining for p53 (B) and Cleaved‐Caspase3 (Cl‐CASP3, (C)) on transverse sections of Ctrl, Sas‐4 ^−/−, Sas‐4 ^−/− 53bp1 ^−/−, and Sas‐4 ^−/− Usp28 ^−/− embryos at E9.5. The area shown encompasses the ventral neural tube and surrounding mesenchyme. Asterisks in (B) denote non‐specific staining of blood cells. Scale bar = 50 μm.

3. D, E

   Quantifications of the p53 (D) and Cl‐CASP3 (E) fluorescence signals shown in (B) and (C), respectively. Three embryos per genotype were used for the quantifications. ***P < 0.001, *P < 0.05 (two‐tailed Student’s t‐test, one‐way ANOVA with Tukey’s multiple comparisons tests gave similar results). Bars represent mean ± s.d. (D) Ctrl: 1.00 ± 0.06 (n = 1,770 cells); Sas‐4 ^−/−: 6.84 ± 0.34 (n = 275), Sas‐4 ^−/− 53bp1 ^−/−: 1.85 ± 0.75 (n = 690), Sas‐4 ^−/− Usp28 ^−/−: 1.50 ± 0.48 (n = 975). (E) Ctrl: 1.00 ± 0.70 (n = 1,690); Sas‐4 ^−/−: 7.88 ± 2.62 (n = 175); Sas‐4 ^−/− 53bp1 ^−/−: 1.43 ± 0.71 (n = 1030); Sas‐4 ^−/− Usp28 ^−/−: 1.57 ± 0.44 (n = 950).

Figure 2. p53 upregulation in Sas‐4 ^−/− embryos is evident by E7.5 and is not due to the onset of 53BP1 or USP28 expression

1. A, B

   Whole‐mount immunostaining and quantification for p53 on Ctrl and Sas‐4 ^−/− embryos at E7.5 (A) and E6.5 (B). Representative sagittal planes are shown and the dotted lines demarcate the epiblast. Quantification of nuclear p53 fluorescence intensity in the epiblast is shown below, normalized to control embryos in the same batch at E7.5 (4 embryos) and at E6.5 (9 embryos). ***P < 0.001 (two‐tailed Student’s t‐test). Bars represent mean ± s.d. Scale bars = 100 μm. (A) Ctrl: 1.04 ± 0.00 (n = 495 cells); Sas‐4 ^−/−: 1.65 ± 0.19 (n = 850). (B) Ctrl: 1.00 ± 0.18 (n = 1,665); Sas‐4 ^−/−: 1.16 ± 0.44 (n = 1,330).

2. C, D

   Immunostaining for 53BP1 and USP28 on sagittal sections of WT embryos at E6.5 (C) or sagittal planes of whole‐mount immunostaining at E5.5 (D). Quantification of the fluorescent signals in the epiblast (Ep) versus visceral endoderm (VE) are shown below. Four embryos per genotype were used for the quantifications. ****P < 0.0001, ***P < 0.001 (two‐tailed Student’s t‐test). Bars represent mean ± s.d. Scale bar = 50 μm. (C) 53BP1 in Ep: 1.00 ± 0.10 (n = 225 cells); 53BP1 in VE: 0.37 ± 0.06 (n = 105); USP28 in Ep: 1.00 ± 0.08 (n = 235); USP28 in VE: 0.17 ± 0.05 (n = 90). (D) 53BP1 in Ep: 1.00 ± 0.24 (n = 225); 53BP1 in VE: 0.68 ± 0.14 (n = 115); USP28 in Ep: 1.00 ± 0.23 (n = 280); USP28 in VE: 0.28 ± 0.00 (n = 110).

Figure 3. Sas‐4 ^−/− mESCs activate the mitotic surveillance pathway

1. A

   Immunostaining for the centrosome marker γ‐tubulin (TUBG) on Ctrl and Sas‐4 ^−/− primary mESCs. The bottom panels are magnifications of the areas marked in the top panels. Scale bars = 20 μm (top) and 10 μm (bottom).

2. B

   Immunostaining and quantification for p53 on Ctrl and Sas‐4 ^−/− primary mESCs in pluripotent and partially differentiated (diff.) conditions. The quantification of p53 fluorescence intensities was normalized to Ctrl (Four independent experiments). **P < 0.01, *P < 0.05 (two‐tailed Student’s t‐test, the comparison of the genotypes in the “Pluripotent” condition was not significant using the one‐way ANOVA with Tukey’s multiple comparisons tests). Bars represent mean ± s.d. Scale bar = 50 μm. Pluripotent Ctrl: 1.00 ± 0.02 (n = 2,030 cells); pluripotent Sas‐4 ^−/−: 1.17 ± 0.10 (n = 2,170); partially diff. Ctrl: 0.62 ± 0.13 (n = 2,745); partially diff. Sas‐4 ^−/−: 1.12 ± 0.24 (n = 2,000).

3. C, D

   Three‐day growth curves of WT and Sas‐4 ^−/− (C) or p53 ^−/− and Sas‐4 ^−/− p53 ^−/− (D) primary mESCs in the indicated conditions starting with 10⁵ cells on Day 0 (Four independent experiments). **P < 0.01, *P < 0.05 (two‐tailed Student’s t‐test). Bars represent mean ± s.d. For details of the measurements, see Materials and Methods (Tables 3 and 4).

Figure 4. Centrioles gradually mature during mouse development

1. A

   Sagittal planes of whole‐mount immunostaining and quantification for TUBG on WT mouse embryos from E3.5 to E6.5. The insets are magnifications of selected centrosomes from the dotted areas denoting the inner cell mass (E3.5) or epiblast (E5.5 and E6.5). Bars represent mean ± s.d (two‐tailed Student’s t‐test or one‐way ANOVA with Tukey’s multiple comparisons tests). Scale bars: E3.5 and E5.5 = 15 μm; E6.5 = 30 μm and 3 μm (insets). E3.5: 88 ± 26% (n = 73 cells from 5 embryos); E5.5: 92 ± 10% (n = 270 from 3 embryos); E6.5: 79 ± 12% (n = 12,082 from 3 embryos).

2. B, C

   Representative gSTED super‐resolution images of immunostaining for TUBG and either CEP164 (B) or ODF2 (C). Whole‐mount staining was used for the CEP164 data as well as for the E3.5 blastocysts for ODF2, whereas staining on sections was used for ODF2 at E5.5 and E6.5. Quantification of the percentage of centrosomes (TUBG) with CEP164 or ODF2 are shown on the right. ****P < 0.0001, ***P < 0.001, *P < 0.05 (two‐tailed Student’s t‐test or one‐way ANOVA with Tukey’s multiple comparisons tests; the ODF2 data between E5.5 and E6.5 was not significant using the latter test). Bars represent mean ± s.d. Scale bar = 0.5 μm. CEP164 at E3.5: 2 ± 2%, (n = 92 centrosomes from 4 embryos); E5.5: 50 ± 2% (n = 249 from 3 embryos); E6.5: 62 ± 0% (n = 856 from 3 embryos). ODF2 at E3.5: 20 ± 9% (n = 108 from 5 embryos); E5.5: 51 ± 4% (n = 294 from 5 embryos); E6.5: 60 ± 2% (n = 1,183 from 4 embryos).

3. D

   Immunostaining and quantification of the area of TUBG and PCNT signals from E5.5 and E6.5 centrosomes in epiblast cells. The separate channels for the marked centriole are shown in the insets. ***P < 0.001, **P < 0.01 (two‐tailed Student’s t‐test). Bars represent mean ± s.d. Scale bar = 2 μm. Quantification of PCNT at E5.5: 0.22 ± 0.01 μm (n = 103 centrosomes from 3 embryos); E6.5: 0.31 ± 0.02 μm (n = 342 from 3 embryos). Quantification of TUBG at E5.5: 0.15 ± 0.02 μm (n = 108 from 4 embryos); E6.5: 0.25 ± 0.01 μm (n = 311 from 3 embryos).

Figure 5. A schematic model depicting the correlation between gradual centriole maturation and centrosome functions during mouse embryonic development

Centrioles first form by de novo biogenesis around E3 and gradually mature, acquire appendages (orange spokes) and recruit PCM (pink circle) to provide a basal body template for cilia and act as MTOCs for mitosis around E7, when the mitotic surveillance pathway is established. The expression of 53BP1 and USP28 precedes the establishment of the pathway and continues until at least E9.5. The embryo development timeline was adapted and modified from (Kojima et al, 2014).

Figure EV2. Ift88 ^−/− cilia mutants do not upregulate p53

1. A, B

   Immunostaining and quantification for p53 on whole‐mount Ctrl and Ift88 ^−/− embryos at E7.5 (A) and E6.5 (B). Mid‐sagittal planes are shown with the dotted lines demarcating the epiblast. Scale bars = 100 μm. The quantification of p53 nuclear fluorescence intensity in the epiblast was normalized to Ctrl embryos in the same batch at E7.5 (A) and E6.5 (B). Bars represent mean ± s.d. (two‐tailed Student’s t‐test). (A) Ctrl: 1.00 ± 0.25 (n = 11,695 cells from 8 embryos); Ift88 ^−/− : 0.86 ± 0.24 (n = 40,815 from 3 embryos) (B) Ctrl: 1.00 ± 0.11 (n = 8,895 from 14 embryos); Ift88 ^−/− : 1.16 ± 0.29 (n = 3,470 from 4 embryos).

Figure EV1. 53bp1 ^−/− and Usp28 ^−/− are null alleles

1. A, B

   Immunostaining and quantifications for 53BP1 (A) or USP28 (B) on sagittal sections of control (Ctrl) and Sas‐4 ^−/− 53bp1 ^−/− (A) or Usp28 ^−/− (B) embryos at E9.5. The signals for the corresponding proteins in the mutants compared to controls are not detectable and close to background staining of secondary antibody‐only negative controls, which were used as baseline for quantification. The cervical or brachial neural tube (the dotted quantified area) and underlying mesenchyme are shown. Asterisks indicate non‐specific staining in blood cells. Three embryos per genotype were used for the quantifications. ***P < 0.001, **P < 0.01 (two‐tailed Student’s t‐test). Bars represent mean ± s.d. Scale bars = 100 μm. (A) Ctrl: 1.00 ± 0.12; 53bp1 ^‐/^‐: 0.23 ± 0.04 (B) Ctrl: 1.00 ± 0.26; Usp28 ^‐/^‐: 0.05 ± 0.02.

Figure EV3. Pluripotency, p53 ^−/− null alleles and the mitotic indices in controls and Sas‐4 ^−/− mutants

1. A

   Immunostaining for NANOG on Ctrl and Sas‐4 ^−/− primary mESCs in pluripotent and partially differentiated conditions. Scale bar = 50 μm.

2. B

   Western blot analysis for p53 and GAPDH loading control on Ctrl, p53 ^−/−, and Sas‐4 ^−/− p53 ^−/− mESC lysates. The numbers below p53 ^−/− and Sas‐4 ^−/− p53 ^−/− indicate the number of base pairs deleted.

3. C–F

   The mitotic index, or percentage of pHH3‐positive cells, of Ctrl and Sas‐4 ^−/− embryo epiblast at E6.5 (n = 8, (C)) and E7.5 (n = 6, (D)) or mESCs (Four independent experiments) in pluripotent or partially differentiated (diff.) conditions (E). The graph in (D) represents our previously published data (Bazzi & Anderson, 2014). (F) Immunostaining and quantification of the cilia marker ARL13B, together with the basal body marker TUBG, in Ctrl mESCs in pluripotent and partially diff. conditions (Two independent experiments). The selected areas in the top panels are magnified in the bottom panels. Scale bars = 10 µm (top) and 1 µm (bottom). **P < 0.01 (two‐tailed Student’s t‐test or one‐way ANOVA with Tukey’s multiple comparisons tests for (E)). Bars represent mean ± s.d. (C) Ctrl: 6 ± 2 (n = 9,545 cells); Sas‐4 ^−/−: 7 ± 2 (n = 5,875). (D) Ctrl: 9 ± 2 (n = 5,150), Sas‐4 ^−/−: 13 ± 1 (n = 4,990); (E) Ctrl pluripotent: 6 ± 1 (n = 2,445), Sas‐4 ^−/− pluripotent: 6 ± 1 (n = 2,330); Ctrl partially diff.: 4 ± 1 (n = 3,015); Sas‐4 ^−/− partially diff.: 7 ± 0 (n = 2,280). (F) Pluripotent mESCs: 4 ± 1 (n = 1,553) Partially diff. mESCs: 8 ± 2 (n = 1,538).