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Fig. 1.
JNK is active in the rearranging endoderm during Xenopus gut elongation. (A-C) Endoderm morphogenesis is depicted in representative sections of the midgut during three stages of gut elongation. At stage 35 (A), the endoderm cells (yellow) of the primitive gut tube have not yet aligned along the apicobasal axis of the gut tube. By stage 40 (B), most of the endoderm cells have become apicobasally aligned. Over subsequent stages, the endoderm cells radially intercalate, as indicated by the black arrows, opening the central lumen, forming the digestive epithelium and driving gut tube elongation. At stage 46 (C), a mature endoderm epithelium lines the fully elongated gut tube. (D-I) Immunohistochemical staining reveals E-cadherin (Ecad, green) and nuclei (blue) or phosphorylated Jun (pJun, red), as indicated. JNK activity is indicated by the accumulation of pJun in the endoderm nuclei at all stages. Scale bars: 50 μm.
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Fig. 2.
JNK is required for gut tube elongation, but not for digestive organ patterning. (A-C) Embryos were exposed to DMSO, SP600125 or Rockout (RO) from stage 35. Compared with the long coiled intestine in stage 46 DMSO controls (A), gut elongation is severely disrupted in embryos exposed to SP600125 (B) and RO (C). Decreased phosphoJun (pJun) levels in stage 46 gut extracts (inset, B) confirm the efficacy of JNK inhibition in the gut by SP600125. The efficacy of Rockout (∼74% reduction in Rho kinase activity) was confirmed using a Rho-kinase Assay Kit (Cyclex; not shown). (D-O) Gut-specific gene expression patterns were assessed by in situ hybridization in developing gut tubes isolated at stage 43 (D-F), 42 (G-L) or 45 (M-O). Appropriate region- and tissue-specific expression of Nkx-2.5 (D-F; mesoderm boundary between stomach and duodenum), Hhex (G-I; liver; G is shown in dorsal view), Pdx (J-L; pancreas and duodenal endoderm) and IFABP (M-O; intestinal endoderm) is evident under all conditions (n=6-17). FG, foregut; L, liver; MG, midgut; P, pancreas.
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Fig. 3.
JNK is required for endoderm cell rearrangements. (A-H) The prospective gut endoderm was injected with two lipophilic dyes (DiI, red; DiO, green) in anterior-posterior (A-P labeling; A-D) or dorsal-ventral (D-V labeling; E-H) orientations at stage 24. Labeled embryos were exposed to DMSO (B,F), SP600125 (C,G) or Rockout, RO (D,H), from stage 35-46; whole guts were dissected to visualize the final longitudinal distribution of each dye (indicated by red or green brackets). Labeled cells become distributed along the axis of the gut tube in DMSO controls (B,F), but fail to rearrange in the presence of SP600125 (C,G) or Rockout (D,H). (I-L′) Deep endoderm cells of the prospective gut tube were injected with DiI (red) at stage 24 to achieve enter labeling as shown (I, stage 37). Labeled embryos were then exposed to DMSO (J,J′), SP600125 (K,K′) or RO (L,L′) from stages 35 to 46, bisected and counterstained with phalloidin (green). In DMSO controls (J,J′), the labeled cells have radially intercalated and are incorporated within the gut epithelium. In the presence of SP600125, labeled cells are confined to a central oreof endoderm (dashed circles in K.K′) or to a radial quadrant of stratified cells (dashed lines in K). In the presence of RO, labeled cells span the entire diameter of the tube (L,L′). (M) The frequency of individual guts with the DiI label contacting only the basement membrane (black), occupying a radial quadrant of the gut tube (blue), spanning the gut diameter (red) or confined only to the center (yellow) after exposure to DMSO, SP600125 or RO. Scale bars: 50 μm.
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Fig. 4.
JNK is required for endoderm cell adhesion. (A-F) Embryos were exposed to DMSO (A,D), SP600125 (B,E) or Rockout (RO; C,F) from stage 35-46 and transverse sections were immunohistochemically stained to reveal E-cadherin as a marker of cell-cell adhesion at basolateral membranes (Ecad, green), atypical protein kinase C as a marker of apical polarity (aPKC, red) and DAPI-stained nuclei (blue) in the developing gut tube. Compared with the mature epithelium found lining the sections of the elongated DMSO control gut, epithelial morphogenesis is severely disrupted in the shortened guts induced by exposure to SP600125 and RO. SP600125-exposed guts display a ring of aPKC (arrows, E) and generally reduced levels of E-cadherin (compare E with D,F), whereas RO guts possess aberrant foci of aPKC throughout the endoderm (arrowheads in C,F). Asterisks in B and E indicate the central core of unintercalated, non-adherent endoderm cells (nnerpopulation). (G) Western blot analyses confirm reduced levels of E-cadherin (Ecad), β-catenin (β-cat) and α-catenin (α-cat) in guts isolated from embryos exposed to SP600125 (S), compared with DMSO (D) and RO (R). By contrast, levels of aPKC are downregulated by exposure to RO, but unaffected by exposure to DMSO or SP600125. ERK, loading control. (H) Masses of loose endoderm cells (arrows) protrude from the guts of embryos exposed to SP600125 from stage 28-45, indicating defective tissue cohesion. (A DMSO control embryo is shown in the inset.) (I-L) Stage 46 guts were dissected from embryos exposed to DMSO (J), SP600125 (K) or Rockout (L) from stage 35 and dissociated into a single cell suspension in calcium- and magnesium-free medium; dissociated cells reaggregate into multicellular clusters upon reintroduction of calcium. The size (area) of cell clusters (arrows) derived from SP600125 guts is significantly smaller than clusters derived from DMSO or RO guts, indicating that SP600125 guts have decreased calcium-dependent (i.e. cadherin-based) cell-cell adhesion. By contrast, clusters derived from RO guts are significantly larger than DMSO or SP600125 clusters (I; n=10-22 clusters per condition). *P<0.01; **P<0.01. Each box plot is displayed as the median surrounded by a box representing the interquartile range; error bars indicate minimum and maximum values. Scale bars: 50 μm.
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Fig. 5.
Morpholino knockdown of JNK1 results in loss of cell-cell adhesion. (A,B,D,E) Embryos were injected with a control morpholino oligonucleotide (Cont MO; A,B,F,G,J,K,N,O) or a morpholino (MO) targeting Xenopus JNK1 (JNK1 MO; D,E,H,I,L,M,P,Q). (C) Knockdown of JNK1 protein and JNK activity (phosphoJun) is evident in extracts from JNK MO-injected (mosaic) guts, compared with Cont MO-injected gut extracts. (B,E) MOs were co-injected with mCherry mRNA as a lineage tracer to confirm gut targeted injection (red). (F-Q) Sections of Cont or JNK MO-injected embryos reveal the localization of atypical protein kinase C and mCherry (aPKC, green; mCh, red; F-I), E-cadherin and laminin (Ecad, green; lam, red; J-M), and integrin-β1 (Int, green, to delineate cell outlines; N-Q) in the gut. Endoderm cells in stage 46 Cont MO-injected embryos (red cells in F-G) undergo normal intercalation and epithelial morphogenesis, as indicated by the single layer of columnar epithelial cells (N-O) with normal E-cadherin (J,K). By contrast, endoderm cells with MO-disrupted JNK1 function (red cells in H,I) do not radially intercalate or form a normal epithelium, as indicated by their irregular cell shapes (P,Q) and reduced levels of E-cadherin (L,M). Asterisks indicate an inner population of endoderm cells. (G,K,O,I,M,Q) Higher magnification images of boxed areas in F,J,N,H,L,P, respectively. Scale bars: 50 μm.
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Fig. S1. JNK is required for the localization of adherens junction components during Xenopus gut morphogenesis. (A-L) Embryos were exposed to DMSO or SP600125 from stages 35 to 46, fixed, sectioned and immunohistochemically processed to reveal E-cadherin (Ecad, green; A-D), the adherens junction components α-catenin (αcat, red; A-F) and β-catenin (βcat, red; G-L), the gut mesodermal marker, smooth-muscle actin (green, as a counterstain; G-J) and DAPI-stained nuclei (blue) in the developing gut tube, as indicated. Asterisks indicate the inner population of endoderm cells. Scale bars: 50 μm.
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Fig. S2. Increasing concentrations of SP600125 and Rockout elicit increasingly short guts, but with distinct changes in tissue architecture. (A-R) Embryos were exposed to DMSO (A), SP600125 (C,E,G,I) or Rockout (K,M,O,Q) from stage 35 through 46, at the concentrations indicated. Transverse sections of a representative embryo exposed to DMSO (B) and each concentration of SP600125 (D,F,H,I) or Rockout (L,N,P,R) were stained to reveal the presence of E-cadherin (green; cell-cell adhesion) and laminin (red; basement membrane). Increasing levels of SP600125 elicit more severe gut elongation defects accompanied by decreasing levels of E-cadherin and a bilaminar endoderm architecture. Increasing levels of Rockout also elicit progressive defects in gut elongation, but E-cadherin levels remain unaffected, except for the appearance of foci of upregulated adhesive contacts (arrowheads).
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Fig. S3. SP600125-induced defects in adhesion precede apoptosis. (A-L) Embryos were exposed to DMSO, SP600125 or Rockout from stage 35-38 (12 hours; A-F) or 35-46 (48 hours; G-L), fixed, sectioned and immunohistochemically processed to reveal E-cadherin (Ecad, green), activated caspase 3 (casp, red) and DAPI-stained nuclei (blue) in the developing gut tube. Compared with DMSO and RO, SP600125 guts exhibit reduced and irregular E-cadherin as early as 12 hours after exposure to the inhibitor (B,E). Caspase-positive cells are normally found only in the stomach (arrow, J), but are evident in the core of SP600125 guts after 48 hours exposure (H,K). Caspase-positive cells are undetectable in the RO gut endoderm (I,L). Asterisks indicate the inner cells in the core of the gut. D-F and J-L are higher magnification images of the boxed regions in A-C and G-I, respectively. Scale bars: 50 μm.
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Fig. S4. Morpholino knockdown of JNK1 disrupts endoderm microtubule (MT) architecture. (A-H) Embryos were co-injected with control morpholino (Cont MO; A-D) or JNK1 morpholino (JNK1 MO; E-H) and mCherry mRNA (as a lineage tracer). Sections of injected embryos (stage 46) reveal the localization of atypical protein kinase C and mCherry (aPKC, green; mCh, red; A,C,E,G) or α-tubulin (green; B,D,F,H) in the gut. Cont MO-injected epithelium (red cells in A,C) exhibits normal MT architecture (B,D). By contrast, in the JNK1 MO-injected population (red cells in E,G), MTs are sparse and disorganized (F,H). Asterisks in E-H indicate an inner population of MO-injected cells. (C,D,G,H) Higher magnification images of boxed regions in A,B,E,F, respectively. Images in A,C,E,G have been reproduced from Fig. 5. Scale bars: 50 μm.
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Fig. S5. Microtubule (MT) stabilization does not phenocopy JNK inhibition. (A-F) Embryos were exposed to DMSO (A) or epothilone B (B) from stage 35 through to stage 46. Transverse sections of a representative embryo exposed to DMSO (C,E) and Epothilone B (D,F) were stained to reveal the presence of α-tubulin (green; MTs), β-catenin (red; membrane) and nuclei (blue). An increased number of M-phase stalled mitotic figures (arrowheads) can be observed in the epothilone-treated gut, but gut elongation is only mildly affected; MT arrays and cell-cell adhesion are normal.
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Fig. S6. JNK does not regulate actin polymerization in the gut. (A-F) Embryos were exposed to DMSO (A-C) or SP600125 (D-F) from stage 35 through stage 46 (48 hours). Whole gut tubes were bisected and stained with phalloidin to visualize the distribution of polymerized F-actin. E and F are higher magnifications of the boxed areas in D. Although actin is disrupted in the inner cell population (asterisks in D,E), cortical actin is still evident in the outer cells (F), and a robust actin belt forms at the apical surface of the abnormal epithelium (arrows, E). (G-X) Embryos were exposed to DMSO (G-L,S-U) or SP600125 (M-R,V-X) for 8 hours. Transverse sections of a representative embryo exposed to DMSO (G-L) or SP600125 (M-R) were stained to reveal the presence of α-tubulin (green; MTs) or β-catenin (red; adhesion), as indicated, or whole gut tubes were bisected and stained with phalloidin to visualize F-actin (S-X). Although the 8-hour exposure to SP600125 has disrupted the parallel bundling of MT arrays and the regular distribution of β-catenin at the membrane, there is no observable difference in cortical actin distribution in either the inner (W) or outer (X) endoderm cells. (J-L,P-R) Higher magnification images of the boxed regions in G-I,M-O, respectively. (T-U,W-X) Higher magnifications of the boxed areas in S and V, respectively. Scale bars: 50 μm.
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