Figure 6.
Figure 6. Genes involved in fatty acid synthesis are required to form the permeability barrier. (A) Schematic outline of some of the genes in the fatty acid biosynthetic and modification pathway. CYP, Cytochrome P450. (B) Confocal images of control embryos and embryos in which the indicated proteins were inhibited by RNAi. (first column) The middle chitin layer was visualized in fixed embryos by staining for chitin and DNA. White asterisks mark extruded polar bodies. (second column) The inner CPG layer was visualized in embryos expressing mCherry::CPG-1. (third column) The presence of the permeability barrier was assessed in embryos expressing mCherry::CPG-2 and a GFP-tagged plasma membrane marker. In control embryos, the permeability barrier prevents diffusion of mCherry::CPG-2 to the embryo surface (open arrowhead). When fatty acid synthesis is inhibited, the permeability barrier is disrupted, and mCherry::CPG-2 fills the entire space between the eggshell and embryo surface (closed arrowheads). (last column) Embryos expressing a GFP-tagged plasma membrane probe were placed in FM4-64 dye to test their permeability. (C) The phenotypic consequences of disrupting the eggshell permeability barrier (cyp-31A2/3(RNAi)), the inner CPG layer (cpg-1/2(RNAi)), and the middle chitin layer (chs-1(RNAi)) were compared by analyzing the percentage of embryos exhibiting each of the indicated phenotypes. Data were pooled from >10 independent imaging sessions for each condition. Plasma membrane adhesion (yellow arrows) was also usually accompanied by cytokinesis failure (59% of cpg-1/2(RNAi) embryos failed cytokinesis). In addition to the quantified phenotypes, chitin layer disruption also led to polyspermy (24% of chs-1(RNAi) embryos were polyspermic). The images of embryos illustrating membrane adhesion and eggshell rupture (white arrowhead) are reproduced from Fig. 3 B. The left image is of an embryo expressing a GFP-tagged plasma membrane marker and mCherry::histone H2B (red) that was placed in FM4-64 dye. Bars, 10 µm. n = number of imaged embryos.

Genes involved in fatty acid synthesis are required to form the permeability barrier. (A) Schematic outline of some of the genes in the fatty acid biosynthetic and modification pathway. CYP, Cytochrome P450. (B) Confocal images of control embryos and embryos in which the indicated proteins were inhibited by RNAi. (first column) The middle chitin layer was visualized in fixed embryos by staining for chitin and DNA. White asterisks mark extruded polar bodies. (second column) The inner CPG layer was visualized in embryos expressing mCherry::CPG-1. (third column) The presence of the permeability barrier was assessed in embryos expressing mCherry::CPG-2 and a GFP-tagged plasma membrane marker. In control embryos, the permeability barrier prevents diffusion of mCherry::CPG-2 to the embryo surface (open arrowhead). When fatty acid synthesis is inhibited, the permeability barrier is disrupted, and mCherry::CPG-2 fills the entire space between the eggshell and embryo surface (closed arrowheads). (last column) Embryos expressing a GFP-tagged plasma membrane probe were placed in FM4-64 dye to test their permeability. (C) The phenotypic consequences of disrupting the eggshell permeability barrier (cyp-31A2/3(RNAi)), the inner CPG layer (cpg-1/2(RNAi)), and the middle chitin layer (chs-1(RNAi)) were compared by analyzing the percentage of embryos exhibiting each of the indicated phenotypes. Data were pooled from >10 independent imaging sessions for each condition. Plasma membrane adhesion (yellow arrows) was also usually accompanied by cytokinesis failure (59% of cpg-1/2(RNAi) embryos failed cytokinesis). In addition to the quantified phenotypes, chitin layer disruption also led to polyspermy (24% of chs-1(RNAi) embryos were polyspermic). The images of embryos illustrating membrane adhesion and eggshell rupture (white arrowhead) are reproduced from Fig. 3 B. The left image is of an embryo expressing a GFP-tagged plasma membrane marker and mCherry::histone H2B (red) that was placed in FM4-64 dye. Bars, 10 µm. n = number of imaged embryos.

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