Fig 2: Abrogation of Dido3 causes chromosome segregation defects and provokes a DNA damage response. (a) Centrosome number was determined by staining for γ-tubulin and centrin (red dots) on cytospin slides of disaggregated Wt/Wt and ΔCT/ΔCT embryos at E7.5. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue background). (b) The incidence of centrosome anomalies in these embryos was quantified as the presence of more than two or less than one centrosome per cell as a percentage of total cells analyzed. Data were obtained from four mice per genotype and analyzed for statistically significant differences using the Fisher exact test. (c) Sections from Wt/Wt and ΔCT/ΔCT embryos at E7.5 were assayed for DNA damage (cells positive for phosphorylated γ-H2AX, brown dots) and apoptosis (TUNEL-positive cells, green dots). Arrowheads indicate areas of positive staining in the epiblast. Nuclei were DAPI-counterstained (blue). DAPI and TUNEL staining were performed on the same sections of a paraffin-embedded embryo, whereas γ-H2AX detection was performed in a different embryo. (d) Sections from Wt/Wt and ΔCT/ΔCT embryos at E7.5 were assayed for proliferation by monitoring BrdU incorporation 1 h post-BrdU injection into pregnant mice, followed by fluorescein isothiocyanate-anti-BrdU staining (green). Nuclei were DAPI stained (blue). Selected areas of the epiblast (indicated by numbers 1 and 3 in Wt/Wt and ΔCT/ΔCT embryos, respectively) and trophoblast (2 and 4 in Wt/Wt and ΔCT/ΔCT embryos, respectively) are shown at higher magnification. (e) p21 and p16 expression levels were assayed by RT-PCR in tissue homogenates of Wt/Wt and ΔCT/ΔCT embryos at E7.5. Data show mean±S.E.M. from three mice per genotype, each in triplicate and normalized to 18S mRNA expression. Statistical significance was calculated using Student's t-test. (f) Representative western blot analysis of Wt/Wt and ΔCT/ΔCT embryo lysates at E7.5 for the active growth-promoting form of retinoblastoma protein phosphorylated at serines 807/811 (pRb), using β-actin as loading control

Fig 3: Relationship between telomere length and meta-TIFs. (A) Percentage of meta-TIFs with detectable (+) or undetectable (−) telomere FISH signals (mean±s.d., n=3). (B) Comparison of relative fluorescence intensity of γ-H2AX IF (red) and telomere FISH (green). (C) Quantification of relative telomeric fluorescence intensity between the γ-H2AX-positive and -negative sister telomeres of a chromosome-type meta-TIF (mean±s.d., n=3). (D) Cyto-centrifuged chromosomes stained with DAPI (blue), γ-H2AX IF (red) and TRF2 or RAP1 IF (green). DAPI, 4′,6-diamidino-2-phenylindole; DDR, DNA damage response; FISH, fluorescence in situ hybridization; γ-H2AX, phosphorylated H2AX; IF, immunofluorescence; meta-TIF, metaphase telomere dysfunction-induced focus; PD, population doubling; Tel, telomere.

Fig 4: Five DDR+ telomeres precede replicative senescence. (A) Chromosome- and chromatid-type meta-TIFs as a percentage of total meta-TIFs (mean±s.d., n=3). Examples of chromatid- and chromosome-type meta-TIFs stained with DAPI (blue), γ-H2AX IF (red) and telomere FISH (green). (B) CO-meta-TIF assay results (mean±s.d., n=3) and examples stained with DAPI (blue), γ-H2AX IF (red) and telomere C-strand PNA FISH (green). (C) Flow cytometry of PI-stained dividing and senescent normal fibroblasts. (D) Calculated number of predicted G1-TIFs. CO, chromosome orientation; DAPI, 4′,6-diamidino-2-phenylindole; DDR, DNA damage response; FISH, fluorescence in situ hybridization; γ-H2AX, phosphorylated H2AX; IF, immunofluorescence; meta-TIF, metaphase telomere dysfunction-induced focus; PD, population doubling; PI, propidium iodide; PNA, peptide nucleic acid.

Fig 5: YTHDC2 localizes to RNA granules.(A-A′′) High-magnification immunofluorescence images of spermatocytes from an adult mouse testis cross-section stained for YTHDC2 (green) DCP1A (red) and DAPI (blue). (B) High-magnification image of an early spermatocyte from a human testis cross-section stained for YTHDC2 (green), γ-H2AX (red) and DAPI (blue). (C) High-magnification image of a late, pachytene-stage spermatocyte from a human testis cross-section stained for YTHDC2 (green), γ-H2AX (red) and DAPI (blue). (D) Immunoprecipitation with α-YTHDC2 from adult wild-type (wt) and Ythdc2-/- mouse testis extracts. Western blots probed with α-YTHDC2, α-MIWI, or α-MSY2. (E) Immunoprecipitation with α-MSY2 and α-IgG from adult wild-type mouse testis extracts. Western blots probed with α-YTHDC2 and α-MSY2. (F) Immunoprecipitation with α-MIWI and α-IgG from adult wild-type mouse testis extracts. Western blots probed with α-YTHDC2 and α-MIWI. (G) Model for action of YTHDC2 with germ granule components in spermatocytes. See also Figure 9—figure supplement 1.