Atrn regulates actomyosin formation and Actin-NMII interaction in zebrafish embryos. (A) Confocal images of phalloidin stained embryos at 50% epiboly, and phalloidin and anti-NMII double stained embryos at 75% epiboly. The mutant embryos were collected at both the same time point and comparable morphological stages compared with wild-type embryos. Embryos were laterally viewed with animal pole to the top and vegetal pole to the bottom. (B) Quantitative measurements of actomyosin band widths in (A) at 75% epiboly stage, which are represented by the widths of Actin and NMII staining in the E-YSL separately. (C) Quantitative measurements of the marginal EVL cell length/width ratios in (A) at both 50% and 75% epiboly stages. Ne, the number of observed embryos; Nc, the number of observed cells. (D) Epiboly defects caused by YSL-injection of atrn-MO. 10 ng std-MO and atrn-MO were used. (E) Epiboly progression in (D) was measured by the average epiboly percentage of the embryos. (F) Atrn associated with less catalytically inactive form of ALKBH4 (ALKBH4-Mut), as revealed by co-IP in HEK293T cells. (G) Co-IP assay shows that endogenous Actin-NMII interaction is decreased in MZatrn mutant embryos. Embryos at 75% epiboly were harvested for lysis and Co-IP. (H) Rescue effects of wild-type and two methylation-deficient actin mRNAs in MZatrn embryos. 100 pg mcherry and different forms of actin mRNA were used for injection. Embryos injected with 100 pg mcherry mRNA were used as negative controls. Embryos were observed at dome and 75% epiboly stages, and shown as lateral views. (I) Statistical results of (H). *, p<0.01. Scale bars: 50 μm in (A); 100 μm in (D, H).
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