Jun N-terminal kinase maintains tissue integrity during cell rearrangement in the gut

Abstract

Tissue elongation is a fundamental morphogenetic process that generates the proper anatomical topology of the body plan and vital organs. In many elongating embryonic structures, tissue lengthening is driven by Rho family GTPase-mediated cell rearrangement. During this dynamic process, the mechanisms that modulate intercellular adhesion to allow individual cells to change position without compromising structural integrity are not well understood. In vertebrates, Jun N-terminal kinase (JNK) is also required for tissue elongation, but the precise cellular role of JNK in this context has remained elusive. Here, we show that JNK activity is indispensable for the rearrangement of endoderm cells that underlies the elongation of the Xenopus gut tube. Whereas Rho kinase is necessary to induce cell intercalation and remodel adhesive contacts, we have found that JNK is required to maintain cell-cell adhesion and establish parallel microtubule arrays; without JNK activity, the reorganizing endoderm dissociates. Depleting polymerized microtubules phenocopies this effect of JNK inhibition on endoderm morphogenesis, consistent with a model in which JNK regulates microtubule architecture to preserve adhesive contacts between rearranging gut cells. Thus, in contrast to Rho kinase, which generates actomyosin-based tension and cell movement, JNK signaling is required to establish microtubule stability and maintain tissue cohesion; both factors are required to achieve proper cell rearrangement and gut extension. This model of gut elongation has implications not only for the etiology of digestive tract defects, but sheds new light on the means by which intra- and intercellular forces are balanced to promote topological change, while preserving structural integrity, in numerous morphogenetic contexts.

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.

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.

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 ‘Center 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 ‘core’ of 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.

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 (‘inner’ population). (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.

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.

Fig. 6.

JNK is required for endoderm microtubule (MT) architecture. (A-R) Embryos were exposed to DMSO (A-C′,J-L), SP600125 (D-F′,M-O) or Rockout (RO; G-I′,P-R) from stage 35 through to stage 40 (A,D,G), 42 (J-R), 43 (B,E,H) or 46 (C,C′,F,F′,I,I′), and transverse sections were stained with α-tubulin (green) to reveal MT architecture, β-catenin (red) to indicate cell-cell adhesive contacts and/or DAPI to show nuclei (blue). (C′,F′,I′) Higher magnification images of the boxed areas in C,F,I, respectively. Apicobasally oriented MT arrays (arrows) are evident in DMSO control guts (A-C′,K), but MT polymerization is disrupted in the presence of SP600125 (D-F′,N), especially in the inner cell population (asterisks, F,F′). Although MT arrays are predominantly apicobasally oriented in RO guts at stage 42 (Q), abnormal foci of MTs eventually form throughout the gut (arrowheads, H-I′). (S-U) Polar coordinate diagrams show the orientation of the MTs in DMSO, SP600125 and RO guts (stage 42). Black bars show the alignment of individual MTs with respect to the apicobasal axis (AB) of the relevant cell, whereas the length of the bar indicates the relative numbers of MTs oriented at that angle. Bars in yellow sectors indicate MTs oriented within 30° of the apicobasal axis, whereas bars in orange sectors indicate MTs that deviated outside of this region. In DMSO (S) and RO (U) guts, most MTs are oriented in parallel (within the yellow sectors); however, the angles of individual MTs are more variable in guts exposed to SP600125 (T). Scale bars: 50 μm.

Fig. 7.

Microtubule polymerization regulates endoderm adhesion. (A-N) Embryos were exposed to DMSO (A-G) or nocodazole (H-N) during the stages prior to gut elongation (stage 35-41), and recovered in normal medium through stage 46. Compared with DMSO controls (A), nocodazole exposure perturbs gut elongation (H). Transverse sections were stained for β-catenin (red, cell-cell adhesion; B,E,I,L) and α-tubulin (green, MTs; C,F,J,M), with merged (red-green) images shown in D,G,K,N. Compared with the mature epithelium found lining the elongated gut tube of DMSO controls (B-G), epithelial morphogenesis is severely disrupted in the shortened guts induced by exposure to nocodazole (I-N). Endoderm cells are deficient in both MTs and β-catenin, and sort into inner (asterisks) and outer populations. Scale bars: 50 μm.

Fig. 8.

Model of the complementary roles of ROCK and JNK in gut elongation. Rho activates actomyosin-based contractility via Rho kinase (ROCK) to generate tension and remodel cadherin-based cell-cell adhesive contacts, leading to the radial reorganization of the gut endoderm (blue arrows). By contrast, JNK signaling stabilizes cell-cell adhesion, either directly and/or via its effects on microtubule architecture (red arrows), to maintain structural integrity during endoderm rearrangement. Both pathways must be coordinated in space and time, within and between cells, to control cell intercalation events in order to achieve anisotropic tissue extension to elongate the gut tube. See text for further details.