Formation of a functional permeability barrier requires passage through anaphase of meiosis II. (A) Confocal images of control (n = 12), chs-1(RNAi) (n = 18), and cpg-1/2(RNAi) (n = 18) embryos expressing a GFP-labeled plasma membrane probe and mCherry::histone H2B (red) that were dissected into FM4-64 dye (illustrated in schematics). (B) Embryos expressing GFP::histone H2B (green) were dissected in FM4-64 dye at different stages between anaphase of meiosis I and anaphase of the first mitotic division. Arrow positions indicate the times when that embryo was exposed to dye, and the color indicates whether it was permeable. White arrows highlight weak FM4-64 staining of the plasma membrane in a partially permeable embryo. (C) Time-lapse confocal images of a cul-2(RNAi) embryo expressing mCherry::CPG-2 and GFP::histone H2B (n = 19). Times are minutes and seconds past anaphase I. (D) Control (top; n = 8) and cul-2(RNAi) (bottom; n = 44) embryos expressing GFP::histone H2B and GFP::γ-tubulin were exposed to FM4-64 dye at different stages to assess eggshell permeability. Arrow positions indicate the time when that embryo was exposed to dye, and the color indicates permeability status. Embryos observed during this experiment whose temporal staging was imprecise are represented by the green and red striped region of the timeline rather than arrows (21 permeable, 2 partially permeable, and 11 impermeable embryos). Bars, 10 µm.