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Figure 6.
A: Schematic of the stages examined during buccopharyngeal perforation. B–G: Frontal views of the buccopharyngeal membrane labeled with E-cadherin in control and JNK inhibitor treated embryos. White arrows point to examples of cells with an increase in cytoplasmic labeling and white arrow heads indicate lower E-cadherin at the membrane. White dots outline the holes or perforation. H–M: Frontal views of the buccopharyngeal membrane labeled with clathrin in control and JNK inhibitor treated embryos. White dots outline the holes or perforation. N–S: Colocalization experiment with phospho-JNK (green) and clathrin (red). N: pJNK1 in green. O: Clathrin in red. P: Merge of pJNK1 and clathrin. Q: Volume rendered image of pJNK and clathrin co-labeling rotated 45 degrees. R: Scatterplot of pixels labeled in green vs. red in the image shown in P. S: Bar graphs of the average correlation coefficients for Pearson's and Mander's colocalization tests. T–X: Frontal views of the entire faces (regular letters) or only the buccopharyngeal membrane at two-fold magnification (prime letters) from representative embryos at stage 40–41. T,T′: Representative control embryo treated with DMSO. U,U′: Representative embryo treated with chlorpromazine. V,V′: Representative embryo treated with Bafilomycin. W,W′: Representative uninjected embryo. X,X′: Embryo injected with CA-JNK. Abbreviations: perf, perforated; cg, cement gland; CA-JNK, constitutively active JNK. Red dots outline the embryonic mouth. |
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Figure 5.
A: Schematic showing the steps in the cell spreading assay. B: 24 hr after explanting epidermis and treating it with DMSO (control). C: 24 hr after explanting epidermis and treating it with the JNK inhibitor. D: Schematic showing the treatment paradigm for the EGTA adhesion assay. E: Control embryo treated with DMSO only. Inset shows E-cadherin labeling confined to edges of cells. F: Representative embryo treated with DMSO followed by EGTA. White arrows point toward regions where the tissue is dissociating. Inset shows internalized E-cadherin. G: Embryo treated with JNK inhibitor alone. Inset shows E-cadherin labeling confined to cell boundaries. H: Embryo treated with JNK inhibitor followed by EGTA. Inset shows E-cadherin largely confined to the cell boundaries. I: The epidermis of a representative embryo showing the cells containing fluorescein labeled Control MO (green) and E-cadherin labeled in red. I′: Corresponding image from I showing only the E-cadherin labeling in red. I′: Corresponding image from I showing only the E-cadherin labeling in black and white. Insets show representative 1–2 cells at 2 × magnification. J: The epidermis of a representative embryo showing the cells containing fluorescein labeled JNK1 MO (green) and E-cadherin labeled in red. J′: Corresponding image from J showing only the E-cadherin labeling in red. J′: Corresponding image from J showing only the E-cadherin labeling in black and white. Insets show representative 1–2 cells at 2 × magnification. Black arrows indicate groups of cells with higher levels of E-cadherin at the membrane. K–L: E-cadherin labeling of epidermis from representative embryos (stage 24–26) that were uninjected (K,K′) or injected with CA-JNK (L,L′). Prime letters are the identical pictures in black and white. Insets show a two-fold magnified image of a cell from the images. White arrows show what appears as puncta of E-cadherin positive bodies within the cytoplasm and arrowheads point to membranes with reduced E-cadherin. Insets show representative 1–2 cells at 2 × magnification. M–N: Clathrin labeling of epidermis from representative embryos (stage 24–26) that were uninjected (M,M′) or injected with CA-JNK (N,N′). Prime letters are the identical pictures in black and white. Insets show representative 1–2 cells at 2 × magnification. White arrows indicate nonspecific staining where fluorescence is observed on top of the cell. |
