Houssin NS , Bharathan NK , Turner SD , Dickinson AJ .

R03 AR065583 NIAMS NIH HHS , R56 DE026024 NIDCR NIH HHS

	Figure 1. A: Schematic showing treatment paradigm. B: Frontal view of a representative Control treated with 1% DMSO. C: Frontal view of a representative embryo treated with JNK inhibitor. D: Frontal view of a representative embryo injected with control morpholino at the 1 cell stage. E: Frontal view of a representative embryo injected with JNK1 morpholino at the 1 cell stage. F: Schematic showing face transplant paradigm. G: Frontal view of a representative control transplant. H: Frontal view of a representative embryo with JNK1 morphant tissue transplanted to its orofacial region. I: Frontal view of a representative embryo with control morpholino injected into D12 blastomere at the 16 cell stage. J: Frontal view of a representative embryo with JNK1 morpholino injected into D12 blastomere at the 16 cell stage. K: A schematic of the JNK inhibitor treatment paradigm and stages examined by histology. L,M: Shows sagittal sections through the buccopharyngeal membrane just before JNK inhibitor treatment at stage 37/38 (two representative images). N,O: Shows sagittal sections through the buccopharyngeal membrane at stage 39 in controls (N) and JNK inhibitor treated (O). P,Q: Shows sagittal sections through the buccopharyngeal membrane at stage 40 in controls (N) and JNK inhibitor treated. perf, perforated; cg, cement gland; inj, injected; MO, morpholino; st, stage. Red dots outline the embryonic mouth.
	Figure 2. A–G: Frontal views of the buccopharyngeal membrane labeled with pJNK1 antibody (green) at three different stages representing before and during perforation. White dots outline “holes” or perforations. H: Sagittal section through the buccopharyngeal membrane and surrounding face showing pJNK (green) and counterstained with propidium iodide. pJNK can also be observed in the two layered epidermis, in the outer epidermis (OE) and inner epidermis (IE). I: Flat mount of the epidermis labeled with pJNK antibody (green). J: The mesoderm labeled with pJNK antibody (green). J′) Corresponding image to J where the pJNK is labeled green and nuclei/DNA labeled with propidium iodide (red). White arrows indicate examples of cells that appear to be undergoing mitosis. K: Magnified image of a mesodermal cell labeled with pJNK antibody (green) (K′) corresponding cell from K labeled with propidium iodide to show DNA (red). K′: Cell corresponding to K showing a merge of pJNK (green) and DNA (red). L: Embryos labeled with pJNK antibody (red) and showing the fluorescein tagged control morpholino (green). L′: Same image as L showing only the pJNK labeling (red). M: Embryos labeled with pJNK antibody (red) and showing the fluorescein tagged JNK1 morpholino (green) M′: Same image as H showing only the pJNK labeling (red). pJNK1, phospho JNK1; st, stage.
	Figure 3. A–D: Shows phospho-histone H3 (ph3; red) marking mitotic cells and labeling of F-actin with phalloidin (green) is used as a counterstain. White arrows indicate the embryonic mouth. A,B: Sagittal section through the middle of the embryo at stage 37/38 after treatment with DMSO/control (A) or JNK inhibitor (B). C,D: Epidermis at stage 37/38 after treatment overnight with DMSO/control (C) or JNK inhibitor (D). E: Schematic showing the morpholinos injected into one cell at the two cell stage and the key where the morpholino can be seen in green and ph3 is labeled in red. F: Dorsal view of an embryo injected as shown in E. G,H: Frontal views of embryos treated with DMSO (control; G) or cell cycle inhibitors hydroxyurea and aphidicolin (HUA; H). Red dots outline the embryonic mouth. cg, cement gland; MO, morpholino; ph3, phospho histone H3; perf, perforated.
	Figure 4. A,B: Lateral view of representative controls (A) and JNK1 morphants (B) where gst1 mRNA (purple) was detected by in situ hybridization. C–F: Live Mitotracker CMXRos labeling (red) after embryos were treated with DMSO (control; C, E) or JNK inhibitor. C,D: Shows the buccopharyngeal membrane. E,F: Shows the epidermis. G,H: Frontal views of the face of representative embryos untreated (G) or treated with hydrogen peroxide (H202; H). Red dots outline the embryonic mouth. I,J: Transverse sections through the face of a representative embryo injected with JNK1 morpholino into one cell at the two cell stage. Tissue containing the fluorescein tagged JNK1 morpholino is green and cleaved caspase-3 labeling (apoptotic marker) is in red. K,L: Frontal views of whole representative faces from embryos treated with DMSO (K) or JNK inhibitor (L). Cleaved caspase-3 labeling is in green and propidium iodide is used a counterstain (red). M,N: Representative images of frontal views of faces of embryos exposed to UV. Control (not irradiated) (M) and irradiated (N) at stage 37/38. Red dots outline the embryonic mouth. perf, perforated; MO, morpholino; BM, buccopharyngeal membrane; CC3, cleaved caspase 3; PI, propidium iodide.
	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.
	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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