Figure 8.
Figure 8. A requirement for PERM-1 and DGTR-1 suggests that an ascaroside may be important for permeability barrier formation. (A) Confocal images of control embryos and embryos in which PERM-1 and DGTR-1 were depleted 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 PERM-1 and DGTR-1 are inhibited, 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. (B) Confocal images of embryos in which PERM-1 and DGTR-1 were inhibited by RNAi in either an rrf-1(+) (left column) or rrf-1(pk1417) mutant background (right column). Embryos expressing a GFP-tagged plasma membrane probe and mCherry::histone H2B (red) were placed in FM4-64 dye to test their permeability. For A and B, n = number of imaged embryos. Bars, 10 µm. (C) Schematics of PERM-1 and DGTR-1 showing the location of domains predicted by BLAST (Basic Local Alignment Search Tool). The TOPCONS program (Stockholm Bioinformatics Center) predicted both proteins to contain two transmembrane domains (2×TM). dTDP, deoxythymidine diphosphate. (D) A putative pathway for ascaroside synthesis in which activated CDP-ascarylose (left side) combines with a long-chain fatty acid–like molecule (right side) to generate ascaroside glycolipid. The reactions proposed to convert CDP-glucose into CDP-ascarylose are based on chemical reactions identified in prokaryotes (Thibodeaux et al., 2007). Points where PERM-1 and DGTR-1 might function in this pathway are indicated.

A requirement for PERM-1 and DGTR-1 suggests that an ascaroside may be important for permeability barrier formation. (A) Confocal images of control embryos and embryos in which PERM-1 and DGTR-1 were depleted 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 PERM-1 and DGTR-1 are inhibited, 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. (B) Confocal images of embryos in which PERM-1 and DGTR-1 were inhibited by RNAi in either an rrf-1(+) (left column) or rrf-1(pk1417) mutant background (right column). Embryos expressing a GFP-tagged plasma membrane probe and mCherry::histone H2B (red) were placed in FM4-64 dye to test their permeability. For A and B, n = number of imaged embryos. Bars, 10 µm. (C) Schematics of PERM-1 and DGTR-1 showing the location of domains predicted by BLAST (Basic Local Alignment Search Tool). The TOPCONS program (Stockholm Bioinformatics Center) predicted both proteins to contain two transmembrane domains (2×TM). dTDP, deoxythymidine diphosphate. (D) A putative pathway for ascaroside synthesis in which activated CDP-ascarylose (left side) combines with a long-chain fatty acid–like molecule (right side) to generate ascaroside glycolipid. The reactions proposed to convert CDP-glucose into CDP-ascarylose are based on chemical reactions identified in prokaryotes (Thibodeaux et al., 2007). Points where PERM-1 and DGTR-1 might function in this pathway are indicated.

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