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Fig. 8 Overexpression of human E736D COL22A1 mRNA causes cell detachment during gastrulation, while the protein is retained in the cytoplasm leading to an increase in ER stress. (A,B) Cells fail to undergo epiboly and detach from the yolk in the E736D COL22A1 mRNA-injected embryo (B) during gastrulation compared with the human WT COL22A1 mRNA-injected embryo (A). (C-D″) Human E736D COL22A1 protein is retained in the cytoplasm during gastrulation, whereas WT COL22A1 is secreted into the extracellular space (arrows). Confocal microscopy analysis of human COL22A1 protein immunofluorescence (green, C,D) and phalloidin staining (red, C′,D′). Blue, nuclear DAPI staining. (E) Aggregation of human mutant COL22A1 in the cytoplasm induces ER stress. qPCR confirms the elevation of ER stress markers hsp5 and bip in human E736D COL22A1 mRNA-injected embryos compared with the human WT mRNA-injected embryos. Expression in the uninjected samples was normalized to 1. *P<0.05, determined using Student's t-test. Error bars represent s.d.