Figure 4: Disrupted SCMC affects zygote cleavage and spindle function.

(a) Embryos were flushed from the oviduct of five SCMC mutant or control females at E1.5 and stained with Hoechest 33342. Size of each blastomere was calculated by the area of central optical section. The embryo was regarded as asymmetric when the ratio of blastomere sizes in one embryo varied by >10%. Left panel showed DIC images with DNA staining from control and SCMC^Null zygotes. Right panel showed percentage (mean±s.e.m.) of asymmetric divisions by using four to five independent samples (Tle6^Null, 4; Floped^Null, 5; Mater^Null, 4). (b) Zygotes were isolated from Tle6^+/− (control) and Tle6^Nullfemales 28–29 h and 32–33 h after hCG, respectively, cultured for 2–3 h to metaphase and stained with FITC-labelled α-tubulin antibody phalloidin labelled with Alexa Fluor 546 (F-actin), Hoechest 33342 and TLE6 antibody. Scale bar, 20 μm. (c) Zygotes from control and Tle6^Null females were stained as in b. Spindle position was determined by the distance (d, mean±s.e.m.) from the centre (red cross) of zygotes (circle) to the central chromosome (blue line) in their spindle (small oval). The zygote number for each group: Tle6^+/− (n=32), Tle6^Null (n=35); Floped^+/− (n=50), Floped^Null (n, 38); Mater^+/− (n=51), Mater^Null (n=42). ***P<0.001 indicates statistically significant differences using Student’s t-test. (d) Zygotes from control and null females were treated as in b to determine relative length (mean±s.e.m.) of spindle at metaphase. Spindle size in control (Tle6^+/−, n=37; Floped^+/−, n=62; Mater^+/−, n=44) and null zygotes (Tle6^Null, n=35; Floped^Null, n=37; Mater^Null, n=41) are reported as ratio (%) of the spindle to cell length. Student’s t-test was used to test the statistical differences between controls and SCMC^Null zygotes. **P<0.01; ***P<0.001. (e) Zygotes from control and Tle6^Null females were cultured to telophase and stained as in b. Scale bar, 20 μm. (f) Two-cell embryos were stained as in b. Scale bar, 20 μm. (g) Zygotes from control and Tle6^Null were microinjected with β5-tubulin mRNA, cultured for 4–8 h and imaged with the UltraVIEW VoX system to detect spindle movement. Images are representative frames from control (Supplementary Movie 2) and Tle6^Null (Supplementary Movie 3) mouse zygotes. Scale bar, 20 μm.