The subcortical maternal complex controls symmetric division of mouse zygotes by

Size: px

Start display at page:

Download "The subcortical maternal complex controls symmetric division of mouse zygotes by"

Sheryl Goodwin
5 years ago
Views:

1 The subcortical maternal complex controls symmetric division of mouse zygotes by regulating F-actin dynamics Xing-Jiang Yu 1,2, Zhaohong Yi 1, Zheng Gao 1,2, Dan-dan Qin 1,2, Yanhua Zhai 1, Xue Chen 1, Yingchun Ou-Yang 1, Zhen-Bo Wang 1, Ping Zheng 3, Min-Sheng Zhu 4, Haibin Wang 1, Qing-Yuan Sun 1, Jurrien Dean 5,6, Lei Li 1,6 1 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing , China. 2 University of Chinese Academy of Sciences, Beijing , China. 3 State Key laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan , China. 4 Model Animal Research Center and MOE Key Laboratory of Model Animal for Disease Study, Nanjing University, Nanjing , China. 5 Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, MD , USA 6 To whom correspondence should be addressed: J.D.( jurriend@helix.nih.gov) or L.L. ( lil@ioz.ac.cn).

Yu et al., page 2 Supplementary Figure 1. Localization of the SCMC and Establishing Tle6 mutant mice. (a) Mouse early embryos were fixed in the oviduct, embedded in paraffin and sectioned (4 m).

2 Yu et al., page 2 Supplementary Figure 1. Localization of the SCMC and Establishing Tle6 mutant mice. (a) Mouse early embryos were fixed in the oviduct, embedded in paraffin and sectioned (4 m). The paraffin sections were stained with rabbit anti-tle6 and imaged with confocal microscopy. Scale bar, 20 m. (b) The serial pictures were from the time lapse imaging of TLE6-eGFP (Supplementary Movie 1). Normal mouse zygotes were microinjected with the mrna of Tle6-eGFP, cultured for 2-3 hr, and imaged with the UltraVIEW VoX confocal imaging system. Scale bar, 20 m. (c) Representative images of the proximity ligation assay (PLA) with rabbit anti-tle6 and -FLOPED antibodies in eggs and different stage embryos. PLA experiments were

3 Yu et al., page 3 performed as in (Fig. 1d). Scale bar, 50 m. (d) Schematic representation of normal and Tle6 null alleles after targeting with positive (PGK-Neo) and negative (MC1Tk) selectable markers in which 2.1 kbp and 4.5 kbp homologous arms (thicker lines) were placed 5' and 3', respectively, to the Neo cassette. 5' and 3' probes outside of the targeting construct were used to confirm correct targeting. PCR genotyping was performed with primers P1 and P2 for the normal allele (499 bp) and P1 and P3 for the null allele (875 bp). Arabic numbers indicate exons (light-blue boxes). Arrows indicate cleavage sites of the Nhe1 restriction enzyme. (e) Southern hybridization of embryonic stem cell DNA detected the normal allele as a 16.9 kbp fragment with either the 5' or the 3' probe. The Tle6 null allele was detected as 9 kbp and 8.2 kbp fragments with the 5' and 3' probes, respectively. (f) PCR genotyping of mouse tail DNA detected the normal allele (499 bp) with P1 and P2 and the null allele (875 bp) with P1 and P3 primers. (g) Eggs were recovered from hormonally stimulated control (n=14) and Tle6 Null (n=8) females hr after hcg administration. The data represent the number of eggs (mean ± s.e.m.). NS, no statistical difference in Student s t-test, >0.05. (h) Zygotes were isolated from control and Tle6 Null females mated with normal males hr after hcg administration and cultured an additional hr. The progression to 2-cell embryos was assessed morphologically. The data represent the percent of 2-cell embryos (mean ± s.e.m., n=2) observed at 2 hr intervals.

4 Yu et al., page 4 Supplementary Figure 2. Spindle formation and early cleavage in the embryos from null females. (a) Zygotes were isolated from control (Floped +/- ) and Floped Null females hr and hr after hcg, respectively, and cultured to mitosis. After fixation, the embryos were stained with FITC labeled mouse anti- -tubulin, Hoechest (DNA) and rabbit anti-floped antibody (magenta), which was used to distinguish zygotes from control and Floped Null females. Scale bar, 20 m. (b) Similar to (a), but zygotes were obtained from control (Mater +/- ) and Mater Null females and stained with rabbit anti-mater antibody. Scale bar, 20 m. (c) The percent (mean ± s.e.m.; n, 3) of zygotes initiating asymmetric division at anaphase in vitro from control and null females was assessed morphologically. (d) The percent (mean ± s.e.m.) of 2-cell embryos (cultured in vitro) with asymmetric cell division from control (n=11) and null females (TLE6 Null, n=3; FLOPED Null and MATER Null, n=4) was assessed morphologically.

5 Yu et al., page 5 Supplementary Figure 3. Formation of the cytoplasmic F-actin meshwork in the null zygotes. (a) Zygotes at different mitotic stage from Tle6 Null females hr after hcg, cultured to different mitotic stages, stained with Alexa Fluor 546 labeling phalloidin and Hoechest 33342, and imaged by cofocal microscope. A Range Indicator in color look-up table (LUTs) was used to depict the fluorescence intensity of F-actin. To examine the cytoplasmic F-actin, the fluorescence signal of cortical F-actin was saturated. White is the unsaturated and red is the saturated F-actin fluorescence. Scale bar, 20 m. (b) Zygotes were isolated from control and Mater Null females hr after hcg, and cultured to mitotic stages. After fixation, the embryos were stained with FITC labeled mouse anti- -tubulin, phalloidin labeled with Alexa Fluor 546 and Hoechst and imaged by cofocal microscope. Scale bar, 50 m. (c) As (b) but with zygotes from Floped Null females. Scale bar, 20 m.

Yu et al., page 6 Supplementary Figure 4. The subcortical F-actin in SCMC null oocytes. (a) Mouse oocytes were recovered from control and Tle6 Null females 12-13 hr after hcg, respectively.

6 Yu et al., page 6 Supplementary Figure 4. The subcortical F-actin in SCMC null oocytes. (a) Mouse oocytes were recovered from control and Tle6 Null females hr after hcg, respectively. After fixation, the oocytes were mixed and stained with mouse anti-pan-actin, rabbit anti-tle6 antibodies and phalloidin labeled with Alexa Fluor 546. TLE6 staining was used to distinguish oocytes from control and Tle6Null females. F-actin was labeled with phalloidin (red) and used a Rainbow Indicator in color LUTs to depict the fluorescence intensity. Scale bar, 20 m. (b) Paired oocytes from control and Tle6 Null females were recovered and stained as in (a). The immunofluorescence intensity (mean ± s.e.m.) of subcortical F-actin was analyzed with ZEN elite 2011 and calculated by using the control as 100%. The number of paired oocytes was 25. NS indicates no statistically significant differences using Student s t-test, >0.05. (c) Eggs from control and Tle6 Null females were microinjected with UtrCH-eGFP mrna, cultured for 7-14 hr and imaged at the GV, Anaphase I and MⅡstages with UltraVIEW VoX confocal system. Enlarged images indicated the thickness of cortical F-actin. Scale bar, 20 m. (d) Bar graph (mean ± s.e.m.) showed the cortical F-actin thickness of oocytes at GV (control, 29; Tle6 Null, 17), Anaphase I (control, 10; Tle6 Null, 12) and MⅡ(control, 11; Tle6 Null, 15) stages from control and Tle6 Null females. NS indicates no statistically significant differences using Student s t-test, p >0.05.

7 Yu et al., page 7 Supplementary Figure 5. The defect of actin in SCMC mutant zygotes. (a) Zygotes at similar developmental stages (interphase) were recovered from control (Mater +/- ) and Mater Null females hr and hr after hcg, respectively. After fixation and incubation with mouse anti-pan-actin, rabbit anti-mater antibodies and phalloidin labeled with Alexa Fluor 546, embryo were imaged by confocal microscopy. MATER staining was used to distinguish zygotes from control and Mater Null females. Scale bar, 20 m. (b) Similar to (a), but the zygotes were obtained from control (Floped +/- ) and Floped Null females and stained with rabbit anti-floped antibody. Scale bar, 20 m. (c) Zygotes from control (Mater +/- ) and Mater Null females were recovered and stained with DNase I labeled with Alexa Fluor 488 (G-actin) and rabbit anti-mater antibody. Scale bar, 20 m. (d) Similar with (c), but zygotes were from control (Floped +/- ) and Floped Null females and were distinguished using anti-floped antibody. Scale bar, 20 m.

8 Yu et al., page 8 Supplementary Figure 6. F-actin regulators in SCMC null oocytes and zygotes. (a) Zygotes and oocytes at similar developmental stages from control and Tle6 Null females were fixed and stained with rabbit anti-arp2 antibody. Scale bar, 10 m. (b) Zygotes at similar developmental stages from control and Tle6 Null females were fixed and stained with rabbit anti-cdc42 antibody. Scale bar, 10 m. (c) Zygotes at similar developmental stages from control and Tle6 Null and Floped Null females and stained with rabbit anti-fmn2 antibody. Scale bar, 10 m. (d) Zygotes at similar developmental stages from control and Tle6 Null and Floped Null females were fixed and stained with rabbit anti-cofilin antibody. Scale bar, 20 m. (e) Immunoblots of egg and zygote lysates at similar developmental stages from control and Tle6 Null female mice were probed with rabbit anti-profilin 1, rabbit anti-arp2, mouse anti-arp3, rabbit anti-cdc42 and rabbit anti-gapdh antibodies. (f) The abundance (mean ± s.e.m.; n=3) of Profilin 1, Arp2, Arp3 and CDC42 in control and Tle6 Null eggs and zygotes was determined by the intensity of the immunoblot bands. The abundance of the protein in control was set as 100%. (g) The abundance (mean ± s.e.m. n=2) of phosphorylated Cofilin-S3 was determined by the intensity of the immunoblot of mitotic zygotes (prophase, metaphase and telophase) from control and Tle6 Null females. The abundance of pcofilin in control at prometaphase was set as 100%. (h) Normal (Norm) and Tle6 Null ovary lysates were immunoprecipitated with Tle6(α-TLE) antibodys. Input, ovary lysates from normal females; Norm, normal ovary lysates; Null, Tle6 Null ovary lysates.

9 Yu et al., page 9 Supplementary Figure 7. Full scans of western blots in main Figures. (a) Full blots of Figure 2c. (b) Full blots of Figure 3c. (c) Full blots of Figure 7a. (d) full blots of Figure 7c. (e) Full blots of Figure 7d.

MII. Supplement Figure 1. CapZ β2. Merge. 250ng. 500ng DIC. Merge. Journal of Cell Science Supplementary Material. GFP-CapZ β2 DNA

MII. Supplement Figure 1. CapZ β2. Merge. 250ng. 500ng DIC. Merge. Journal of Cell Science Supplementary Material. GFP-CapZ β2 DNA A GV GVBD MI DNA CapZ β2 CapZ β2 Merge B DIC GFP-CapZ β2 Merge CapZ β2-gfp 250ng 500ng Supplement Figure 1. MII A early MI late MI Control RNAi CapZαβ DNA Actin Tubulin B Phalloidin Intensity(A.U.) n=10