Figure 6: The SCMC is required for the assembly of subcortical F-actin in mouse zygotes.

(a) Mouse zygotes at similar developmental stages (interphase) were recovered from control and Tle6^Null females 28–29 h and 32–33 h after hCG, respectively. After fixation, the embryos were mixed and stained with mouse anti-pan-actin, rabbit anti-TLE6 antibodies and phalloidin labelled with Alexa Fluor 546. TLE6 staining was used to distinguish zygotes from control and Tle6^Null females. F-actin was labelled with phalloidin (red) and used a Rainbow Indicator in colour LUTs to depict the intensity of fluorescence, which was shown in the inserted picture. Scale bar, 20 μm. (b) Paired zygotes from control (heterozygote) and null (Tle6^Null, Floped^Null or Mater^Null) females were stained with fluorescently labelled phalloidin and specific antibodies to TLE6, FLOPED and MATER, which were used to separate the zygotes from control or null females. The immunofluorescence intensity of subcortical F-actin was analyzed with ZEN lite 2011. The intensity of subcortical F-actin in controls was set as 100%. The number of paired embryos was 47 for Tle6, 22 for Floped and 46 for Mater. Error bars represents s.e.m. *P<0.05; ***P<0.001 indicates statistically significant differences using Student’s t-test. (c) Zygotes from control and Tle6^Null females were microinjected with UtrCH–eGFP mRNA, cultured for 4–5 h and imaged with UltraVIEW VoX confocal system at the pronuclear stage. Enlarged images (X3) of the control and Tle6^Null zygotes indicated the thickness of cortical F-actin. The red bar showed the thickness of cortical F-actin. Scale bar, 20 μm. (d) Bar graph (mean±s.e.m.) of cortical F-actin thickness at mitotic zygotes from control and Tle6^Null females. Embryo number was 25 (control) or 20 (Tle6^Null). ***P<0.001 indicates statistically significant differences using Student’s t-test. (e) Mouse zygotes from control and Tle6^Null females were recovered as in a and stained with DNase I labelled with Alexa Fluor 488 (G-actin, green), phalloidin labelled with Alexa Fluor 546 (F-actin, red) and rabbit anti-TLE6 antibody (TLE6, magenta). G-actin was used as a Rainbow Indicator to depict the intensity of fluorescence. Scale bar, 20 μm.