Fig. 8

Formation of sub-nuclear structures in post-ZGA embryos is compromised by Rad21 depletion. Rad21-depleted (Rad21 KD) and wild-type stage-matched control embryos at 4.5 hpf were fixed and stained with the indicated antibodies. For all images, nuclei were counterstained with Hoescht. Scale bar: 10 μm. (A,A′) Nucleolin staining (green) in wild type is shown in a field of cells (A) and in a z-stack maximum projection of a single representative nucleus (A′), and indicates the presence of normal nucleoli. (B,B′) Nucleolin staining (green) in Rad21-depleted embryos is shown in a field of cells (B) and in a z-stack maximum projection (B′), and indicates nucleolar dispersion following abrogation of Rad21. (C) Quantification of the area of nucleolin relative to the size of the nucleus in stage-matched wild-type embryos compared with Rad21-depleted embryos at 4.5 hpf (see Materials and methods; n=6 for both conditions) shows that nucleoli fragmentation is significant. Around 200 nuclei for each condition were imaged and analyzed. (D,D′) Staining for the elongating form of RNA polymerase II (p-Ser2-RNAPII) (green) in wild type is shown in a field of cells (D) and in a z-stack maximum projection of a single representative nucleus (D′), and indicates clustering of RNAPII into foci. (E,E′) p-Ser2-RNAPII staining (green) in Rad21-depleted 4.5 hpf embryos is shown in a field of cells (E) and in a z-stack maximum projection (E′), and shows disruption of RNAPII foci upon Rad21 depletion. (F) Quantification of RNAPII foci relative to the size of the nucleus shows statistically significant disruption of RNAPII clustering in Rad21-depleted embryos compared with controls (see Materials and methods; n=6 for both conditions). Around 200 nuclei for each condition were imaged and analyzed. All P values were calculated by applying a t-test with an unpaired fit and assuming a parametric distribution.