Data Supplement
- Supporting Information - Materials and Methods, Table S1 Primers Information and References
- Figure S1 - Cell viability, alkaline phosphatase activity and Gata4 expression of ex vivo guts. (a-g) Small intestine from E13.5 embryos were isolated and cultured to form ex vivo guts, and stained with trypan blue at 3d (a), 5d (b), 7d (c), and 10d (d). (e-f) Magnified image of a selected area in panel c-d. Scale bars are 400 μm in (a-d), and 200 μm in (e-f). (g) Alkaline phosphatase activity of ex vivo guts at 0d, 3d, 5d, 7d, and 10d. Mouse tails from E13.5 embryo were used as the negative control (NC). (f) Ex vivo guts from embryonic distal or proximal small intestine were cultured at 7d. Relative mRNA expression of Gata4 was tested using RT-qPCR. Data are represented as mean ± SEM. N=4. *: P < 0.05 versus distal small intestine. **: P < 0.01 versus 0d.
- Figure S2 - Representative images of negative controls in immunofluorescent staining. Sections were permeabilized, incubated with 5% goat serum overnight at 4°C, and incubated with respective fluorescent secondary antibodies for 2h at room temperature. Immunofluorescence was imaged using a fluorescence microscope. Scale bar is 200 μm.
- Figure S3 - Interstitial cells of Cajal and Foxl1-expressing mesenchymal cells in ex vivo guts. Ex vivo guts were cultured for 7 days. (a-b) Relative C-kit and Foxl1 mRNA in ex vivo guts (EVG). mRNA from intestine and fat tissue of adult mice was used as a positive control (PC) and negative control (NC), respectively. (c) The relative contraction frequencies of ex vivo guts were treated with botulinum neurotoxin for 0, 6, 12 and 24h. Data are represented as mean ± SEM. Each ex vivo gut from a dedicated mouse was view as an experiment unit, n=5. *: P < 0.05 versus 0h. **: P < 0.01 versus 0h.
- Figure S4 - Effects of Wnt, BMP and Notch signaling on intestinal differentiation of ex vivo guts. Small intestine from E13.5 embryos were isolated and cultured in medium containing 100 nM LDN-193189 (BMP signaling inhibitor; a), 10 μM DAPT (Notch signaling inhibitor; b), or 100 ng/ml Wnt 3a plus 500 ng/ml R-spondin1 (Wnt signaling activator; c). Relative mRNA expressions were tested using RT-qPCR. Data are represented as mean ± SEM. N=4. *: P < 0.05 versus CON. **: P < 0.01 versus CON.
Data Supplement
Data Supplement
Data Supplement
Data Supplement
Data Supplement
Data Supplement
Article Figures & Data
Figures
- Figure 1.
Inverted phase contrast images of ex vivo guts. Small intestine from E13.5 embryos were isolated, dissected and cultured to form ex vivo guts at 3 days (a), 4 days (b), 5 days (c), 6 days (d), 7 days (e) and 10 days (f). Scale bar is 250 µm.
- Figure 2.
Haematoxylin and eosin staining images of ex vivo gut cross-sections. Ex vivo guts were collected at day 0 (a), and after 3 days (b), 4 days (c), 5 days (d), 7 days (e) and 10 days (f) of culture, fixed in 4% paraformaldehyde and embedded in OCT, cut into 5 µm section and then stained by haematoxylin and eosin. Scale bar is 125 µm.
- Figure 3.
Localization and distribution of cell differentiation markers in ex vivo guts 7 days post-incubation. Ex vivo guts were cultured for 7 days, fixed and embedded in OCT. The cryosections were examined by Alcian blue staining (a) and immunofluorescence staining (b–f). (a) Goblet cells locate on the surface layer of ex vivo guts shown by Alcian blue staining (black arrows); (b) Paneth cells (white arrows, lysozyme); (c) enteroendocrine cells (chromogranin A staining); (d) columnar epithelium (cytokeratin 8); (e) intestinal transcript factor CDX2; (f) fibroblasts (vimentin), smooth muscle cells (smooth muscle actin) and myofibroblasts (co-immunostaining of smooth muscle actin and vimentin). DAPI was used as nuclei stain. Scale bar is 200 µm in (a–c) and 400 µm in (d–f).
- Figure 4.
Migration and reorganization of ex vivo guts. Lgr5mT/mG ex vivo guts were obtained by crossing Lgr5-EGFP-IRES-creERT2 mice with ROSAmT/mG mice. Lgr5mT/mG ex vivo guts were induced by 250 µm 4-hydroxytamoxifen in the first 3 days of culture to mark Lgr5-derived cells into green fluorescence. The fluorescent signal at the surface of ex vivo guts was observed at day 0 (a), and after 3 days (b), 6 days (c) and 9 days (d), by confocal microscope. Columnar epithelium at 7 days is shown by immunostaining for Cytokeratin 8 (e). The dashed lines indicate the contour of the original gut body. Arrows point to Lgr5-derived cell clusters. Arrowhead points to cells across the border between original gut body and the cells spreading from the gut body. DAPI was used as a nuclear stain. Scale bar is 200 µm.
- Figure 5.
Development of Lgr5 progeny cells and cell signalling in ex vivo guts. (a) Lgr5mT/mG ex vivo guts were obtained by crossing Lgr5-EGFP-IRES-creERT2 mice with ROSAmT/mG mice. Lgr5mT/mG ex vivo guts were induced by 250 μm 4-hydroxytamoxifen in the first 3 days of culture to mark Lgr5-derived cells by the presence of green fluorescence. The fluorescent signal under ex vivo guts (view from the bottom of Petri dish) was observed at 1, 3, 5, 7, 9 and 10 days by fluorescent inverted microscope. (b) Infographic of morphogenesis and migration in ex vivo guts. Cross-section view is above and the top view is below.
- Figure 6.
Differentiation of WT and AMPKα1 KO ex vivo guts. (a) Goblet cells positive ex vivo guts at 7 days. The ex vivo gut cross-sections were stained with Alcian blue, then goblet-positive and goblet-negative ex vivo guts were counted. A total of 70 ex vivo guts were used. (b) The mRNA expression of gut epithelial differentiation markers. (c) The contraction frequencies of ex vivo guts at 7 days were counted per minute. n = 10. (d) Immunostaining of smooth muscle actin for ex vivo guts at 3, 6 and 9 days to identify mesenchymal cells. DAPI was used as a nuclear stain. Scale bar is 200 µm. Data are represented as mean ± s.e.m. *p < 0.05; **p < 0.01.
- Figure 7.
Expression of transcription factors and epithelial cells in WT and AMPKα1 KO ex vivo guts. (a) Ex vivo guts were cross-sectioned at 3, 6 and 9 days. The CDX2 transcription factor and epithelial cells were identified by immunostaining for Cytokeratin 8 and CDX2. Arrows point to CDX2-positive cells in the epithelial layer. DAPI was used as a nuclear stain. Scale bar is 200 μm. (b) mRNA levels of transcription factors in WT and AMPK KO ex vivo guts. Data are represented as mean ± s.e.m. *p < 0.05; **p < 0.01.
- Figure 8.
Epithelial junctions and proliferating and apoptotic cells of WT and AMPKα1 KO ex vivo guts. (a) Ex vivo guts were cross-sectioned at 3, 6 and 9 days. Junctions are determined by immunostaining for E-cadherin. Arrow points to the staining pattern in the lumen. (b) Adherens junction and proliferative cells were visualized by immunostaining for β-catenin and Ki67. DAPI was used as a nuclear stain. (c) Apoptotic cells of ex vivo guts at 7 days were visualized by TUNEL staining. Scale bar is 200 µm. (d) The mRNA expression of apoptotic markers of ex vivo guts at 7 days. Data are represented as mean ± s.e.m. *p < 0.05.
April 2018
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