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Figure 1. Changes in endoderm cell shape and polarity precede elongation in the Xenopus primitive gut tube. Tadpoles were sectioned and immunohistochemically stained to reveal cell membranes (E-cadherin, green), the basement membrane (laminin, red), and nuclei (DAPI [4 prime ,6-diamidine-2-phenylidole-dihydrochloride], blue). The plane of section is indicated in the yellow embryo diagrams (adapted from Nieuwkoop and Faber, [1994]), with stage 32-40 in lateral view and stage 41 in ventral view. Low magnification (00; A,C,E,G) and high magnification (00; B,D,F,H) views of each section are shown. Scale bar = 100 mu M. Brackets in E indicate the region shown in higher magnification in F. The position of the archenteron is indicated by white arrowheads. The color-coded legend refers to the diagrams in B prime , D prime , F prime , H prime that highlight representative endoderm cells with nuclei indicated by circles. A-B prime : Before gut tube elongation (stage 32), most of the endoderm cells are rounded and unpolarized. C-D prime : At stage 37/38, the most basal endoderm cells (arrows, D; blue cells in D prime ) adopt a radial orientation and a polarized morphology. E-F prime : At stage 39-40, deeper endoderm cells (arrows, F; purple cells in F prime ) become polarized and radially oriented. G-H prime : At stage 41, more central endoderm cells are polarized and radially oriented (arrows, H; purple cells in H prime ). fg, foregut; hg, hindgut; mg, midgut.
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Figure 2. Changes in endoderm architecture during elongation and epithelial morphogenesis in the Xenopus primitive gut tube. Tadpoles were sectioned and immunohistochemically stained to reveal cell membranes (E-cadherin, green), the basement membrane (laminin, red), and nuclei (DAPI [4 prime ,6-diamidine-2-phenylidole-dihydrochloride], blue). The plane of section is indicated in the yellow embryo diagrams [adapted from Nieuwkoop and Faber ([1994])]. Low magnification (00; A,C,E,G) and high magnification (00; B,D,F,H) views of each section are shown. Scale bar = 100 mu M. Brackets in G indicate the region shown in higher magnification in H. The position of the gut lumen is indicated by white arrowheads. Color coding and circles in B prime , D prime , F prime and H prime highlights representative endoderm cells as in Figure 1. A-B prime : As the gut lumen begins to expand (stage 42), all endoderm cells are radially oriented and the tissue architecture is stratified columnar, with cells arranged in four to five concentric layers. C-D prime : At stage 43-44, radial intercalation has decreased the thickness and number of cell layers in the gut wall, so that only two to three layers of cells line the PGT. Some cells (green cells in D prime ) span the gut wall from the lumen to the basement membrane. E-F prime : By stage 46, epithelial morphogenesis is almost complete; a single-layered, pseudostratified epithelium now lines the narrowed gut tube. G-H prime : Endoderm cells are also interdigitated with anterior and posterior neighbors within the gut walls (stage 41 shown). I: The average ratio of endoderm cell length to width (L:W ratio) is shown for successive stages 32-46, with standard deviations indicated by error bars. duod, duodenum; mg, midgut; st, stomach.
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Figure 6. Myosin II is expressed in the developing gut tube and is required for gut elongation. A-H: Immunohistochemical staining for Myosin heavy chain IIB (MHC-B, red; A-C,E-G) and E-cadherin (Ecad, green; A,C,D,E,G,H) in transverse sections through the midgut (stage 40-41, A-D; stage 45, E-H) shows that MHC-B is expressed in the endoderm during gut morphogenesis. Before gut elongation, MHC-B is down-regulated in the central core of the gut tube (arrows, A), and is enriched at cell membranes (B), where it colocalizes with E-cadherin (D), particularly at the junctions between intercalating cells (arrows, B-D). After lumen (*) formation, MHC-B becomes enriched at the apical membrane (arrowheads in E) of the developing epithelium (G), and remains colocalized with E-cadherin at the junctions between intercalating cells (arrows, F,H). A,E, 00; B-D,G, 00; F,H, 00. I,J: Compared with dimethyl sulfoxide (DMSO) controls (I), chronic (J; stage 32-46) or limited (K; stage 41-44) exposure to Blebbistatin, a small molecule inhibitor of Myosin II activity, induces severe gut tube elongation defects.
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Figure 7. Rho-associated kinase and Myosin II are required for endoderm cell shape change, gut lumen formation and epithelial morphogenesis. Immunohistochemical staining for E-cadherin (Ecad, green) and laminin (lam, red) as shown in A-I, or E-cadherin (green) and Myosin heavy chain IIB (MHC-B, red) as shown in J-L, was performed on transverse (A-C; stage 41) or frontal (D-I; stage 46) sections through the midgut. Embryos were treated with dimethyl sulfoxide (DMSO; A,D,E,J), a Rho kinase inhibitor (Rockout; B,F,G,K,L), or a Myosin II inhibitor (Blebbistatin; C,H,I) from stage 32. The elongation, radial orientation, and polarization of the endoderm cells occurs normally in control, DMSO-treated embryos (arrows, A). However, in embryos exposed to Rockout or Blebbistatin, endoderm cells are more rounded and not polarized (arrows, B,C). At later stages, Rho kinase-deficient (F,G) or Myosin II-inhibited (H,I) embryos exhibit a thickened gut wall and disorganized epithelial tissue architecture in their shortened gut tubes. The gut lumen (arrowheads, D,F,H) does not expand when Rho kinase (F) or Myosin II (H) activity is inhibited. Moreover, accumulations of E-cadherin can be observed between the layers of unintercalated cells (long arrows, G,I), and centered among cells with abnormal polarity forming rosette-like clusters in the gut wall (short arrows, G,I). Finally, compared with control embryos (J), MHC-B colocalizes abnormally with E-cadherin in the disorganized epithelium of Rho kinase-deficient embryos (K,L). Scale bar = 100 mu M.
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