Formation of the inner CPG layer requires prior deposition of the chitin layer. (A, top) Immunofluorescence images of embryos stained with a chitin-binding probe (white) and for DNA. (bottom) Confocal images of embryos expressing mCherry::CPG-1. (B) Confocal images of cpg-1/2(RNAi) (top; n = 23) and chs-1(RNAi) (bottom; n = 26) embryos expressing a GFP-labeled plasma membrane probe and mCherry::histone H2B. Locations where the plasma membrane is able to separate from the eggshell (scallops; yellow arrows), a failed attempt at cytokinesis (white arrow) and a point where the eggshell lost integrity (rupture; white arrowheads) are marked. Times are minutes and seconds after the first frame. (C) Transmission electron micrographs of high-pressure frozen embryos. (right) Pseudocolored micrographs illustrate the location of the vitelline, chitin, and CPG layers. The mitotic control image is reproduced from Fig. 1 A for comparison. Bars: (A and B) 10 µm; (C) 200 nm. n = number of imaged embryos.