mm-wnt2b  (Advanced Cell Diagnostics Inc)

Journal: Nature Communications

Article Title: Single cell and genetic analyses reveal conserved populations and signaling mechanisms of gastrointestinal stromal niches

doi: 10.1038/s41467-019-14058-5

Figure Lengend Snippet: a 3D images of pericyte-like stromal cells surrounding gastrointestinal epithelial cells are visualized by lineage tracing in Ng2-Cre ; Rosa26 +/mTmG mice. b smFISH in Ng2-Cre ; Rosa26 +/tdTomato mice shows the expression of Wnt2b (green puncta) and Wnt4 (blue puncta) in gastrointestinal pericyte-like stromal cells (tdTomato-red). c Quantitative RT-PCRs shows significantly increased levels of Wnt2b and Wnt4 expression in Ng2-tdTomato positive stomach and intestinal stromal cells compared to Ng2-tdTomato negative cells ( n = 3 per group). d The intestinal length is significantly reduced in Ng2-Cre ; Wls fl/fl mice compared to the controls ( n = 5 per group). e Immunofluorescence staining (IF) of BrdU and Lysozyme C (a Paneth cell marker) shows no defects in intestinal proliferation but a mildly decreased number of Paneth cells in Ng2-Cre ; Wls fl/fl mice compared to the controls ( n = 4 per group). f IF of OLFM4 in antrum and ileum shows significantly decreased numbers of OLFM4+ gastrointestinal stem cells in in Ng2-Cre ; Wls fl/fl mice compared to the controls ( n = 4 per group). Scale bars indicate 20 μm. g smFISH of Lgr5 in the antrum and ileum. Magenta dots indicate Lgr5 transcripts. The number of Lgr5 transcripts was significantly reduced in Ng2-Cre ; Wls fl/fl mice compared to the controls. Scale bars indicate 20 μm. *P < 0.05, * *P < 0.01, *** P < 0.001, **** P < 0.0001. P values were determined using nonparametric unpaired Student’s t test. Values are mean ± SEM. Each n means biologically independent animals and experiments.

Article Snippet: The RNAscope Probe (ACDBio)-Mm- Wnt2b (405031-C2), Wnt4 (401101-C2), Wnt2 (313601), Foxl1 (407401), Rspo3 (402011-C3), Rspo1 (479591-C2), Lgr5 (312171-C3), or Shh (314361-C2) was used for single-molecule FISH.

Techniques: Expressing, Immunofluorescence, Staining, Marker

Journal: Nature Communications

Article Title: Single cell and genetic analyses reveal conserved populations and signaling mechanisms of gastrointestinal stromal niches

doi: 10.1038/s41467-019-14058-5

Figure Lengend Snippet: a Schematic diagram for whole body irradiation and dissection at postnatal day 32 (P32). b smFISH of Wnt2b (Green) in Ng2-Cre ; Rosa26 +/tdTomato mice 48 h after whole body irradiation (9 Gy) shows their significantly increased expression compared to the non-irradiation controls ( n = 3 per group, 2–4 images per mice were counted for Wnt2b expression). c Quantitative RT-PCR shows increased levels of Wnt2b and Wnt4 expression in Ng2-Tdtomato positive cells after 9 Gy irradiation, compared to non-irradiated Ng2-tdTomato positive cells ( n = 3 per group). d Timeline of whole body irradiation. e Body weight change of mice after irradiation ( n = 6 per group). f Survival curve during 10 days after 9 Gy of irradiation ( n = 7 per group). g H&E staining (left), and IF of BrdU (green) and SOX9 (magenta) show a significantly decreased level of gastric epithelial proliferation (BrdU+ cells/gland) and decreased number of Sox9 progenitor cells (Sox9+ cells/gland) in antrum of Ng2-Cre ; Wls fl/fl mice compared to the controls ( n = 3 per group). h IF of CD44 (green) and OLFM4 (magenta) show a significantly decreased number of CD44+ and OLFM4+ progenitors and stem cells, respectively, in the antrum of Ng2-Cre ; Wls fl/fl mice compared to the controls ( n = 3 per group). i H&E staining (left), and IF of BrdU (magenta) and αSMA (green) show a significantly decreased number of BrdU+ cells in the ileum of Ng2-Cre ; Wls fl/fl mice compared to the controls ( n = 3 per group). j IF of CD44 (green) and OLFM4 (magenta) show a significantly decreased number of CD44+ and OLFM4+ progenitors and stem cells, respectively, in the ileum of Ng2-Cre ; Wls fl/fl mice compared to the controls ( n = 3–4 per group). For all, *P < 0.05, * *P < 0.01, *** P < 0.001, * **P < 0.001, * ***P < 0.0001. P values were determined using nonparametric unpaired Student’s t test. Scale bars indicate 20 μm. Values are mean ± SEM. Each n means biologically independent animals and experiments.

Article Snippet: The RNAscope Probe (ACDBio)-Mm- Wnt2b (405031-C2), Wnt4 (401101-C2), Wnt2 (313601), Foxl1 (407401), Rspo3 (402011-C3), Rspo1 (479591-C2), Lgr5 (312171-C3), or Shh (314361-C2) was used for single-molecule FISH.

Techniques: Irradiation, Dissection, Expressing, Quantitative RT-PCR, Staining

Journal: Nature Communications

Article Title: Single cell and genetic analyses reveal conserved populations and signaling mechanisms of gastrointestinal stromal niches

doi: 10.1038/s41467-019-14058-5

Figure Lengend Snippet: a GSEA plot demonstrates enrichment of the Hh signaling pathway in conserved pericyte-like gastrointestinal stromal cells compared to distinct populations. b IF of BrdU and SOX9 in the stomach antrum. The number of SOX9+ and BrdU+ stomach progenitors are significantly increased in Ng2-Cre ; Rosa26 +/SmoM2 mice compared to the controls ( n = 3 per group). c IF of PCNA and αSMA in the intestine. The number of PCNA+ progenitors is significantly increased in Ng2-Cre ; Rosa26 +/SmoM2 mice compared to the controls ( n = 3 per group), *P < 0.05. Mutant pups show a dilated small intestine (Right bottom panel). d IF of Lysozyme C (LyzC, right panel) and Alcian blue staining (left panel) shows increased numbers of intestinal Paneth cells and goblet cells, respectively, in Ng2-Cre ; Rosa26 +/SmoM2 mice compared to the controls. *P < 0.05. Scale bars indicate 50 μm. e , f smFISH combined with differential interference contrast (DIC) imaging demonstrates significantly increased levels of Wnt2b expression (magenta puncta) in gastrointestinal pericyte-like stromal cells of Ng2-Cre ; Rosa26 +/SmoM2 mice compared to the controls ( n = 3 per group). Scale bars indicate 20 μm, *P < 0.05, * *P < 0.01, * **P < 0.001, * ***P < 0.0001. P values were determined using nonparametric unpaired Student’s t test. Values are mean ± SEM. Each n means biologically independent animals and experiments.

Article Snippet: The RNAscope Probe (ACDBio)-Mm- Wnt2b (405031-C2), Wnt4 (401101-C2), Wnt2 (313601), Foxl1 (407401), Rspo3 (402011-C3), Rspo1 (479591-C2), Lgr5 (312171-C3), or Shh (314361-C2) was used for single-molecule FISH.

Techniques: Mutagenesis, Staining, Imaging, Expressing

Journal: Nature Communications

Article Title: Single cell and genetic analyses reveal conserved populations and signaling mechanisms of gastrointestinal stromal niches

doi: 10.1038/s41467-019-14058-5

Figure Lengend Snippet: a Enrichment network. Functional profiling of genes upregulated > twofold in DKO stomach and intestine vs. control. Left half circle: significance of gene set enrichment in stomach. Right half circle: significance of gene set enrichment in intestine. Analysis revealed that the majority of pathways are commonly enriched in both mutant stomach and intestine. Key genes sets: Hh signaling, Wnt signaling, BMP signaling, stem cell development, muscle development, cell fate commitment, angiogenesis, hindgut development (intestine only), response to mechanical stimulus (stomach only). b Correlation heatmap of H3K27ac ChIP-Seq signal at all SE regions identified from DKO stomach and intestine. Plot shows high within-group correlation, and relatively high inter-group correlation, indicating overlapping SEs in the two tissue types. c MA plot of H3K27ac ChIP-Seq signal at SE regions in DKO stomach and intestine confirmed findings in the correlation heatmap. Totally, 538 SE regions were common (fold-enrichment between the two tissues < 1.5). Sixteen regions, mapped to 14 unique genes were tissue-specific (fold enrichment > 2). d Signals from GLI2, H2K27ac, and H3K36me3 ChIP-Seq experiments show examples of intestine ( Hoxc8 )- and stomach ( Nr2f1 )-specific regions regulated by GLI2. e Gli2 ChIP-Seq in DKO stomach and intestine identified over 1100 overlapping regions. f KEGG pathway analysis performed on genes associated with the 1120 overlapping Gli2 peaks. Significant enrichment was identified in Hh, TGF-beta, and Wnt signaling pathways. g Signals from GLI2, H2K27ac, and H3K36me3 ChIP-Seq experiments show examples of Hh signaling ( Ptch1 ) and Wnt ligand ( Wnt2b , Wnt9a ) genes regulated by GLI2. All genes are within SE regions, and are bound by GLI2. h , i Validation of GLI2 activation of luciferase reporter fragments generated from GLI2 peak regions near ( h ) Wnt2b and ( i ) Wnt9a loci. n = 3, Values are mean ± SEM. *P < 0.05. P values were determined using nonparametric unpaired Student’s t test. Each n means biologically independent animals and experiments.

Article Snippet: The RNAscope Probe (ACDBio)-Mm- Wnt2b (405031-C2), Wnt4 (401101-C2), Wnt2 (313601), Foxl1 (407401), Rspo3 (402011-C3), Rspo1 (479591-C2), Lgr5 (312171-C3), or Shh (314361-C2) was used for single-molecule FISH.

Techniques: Functional Assay, Mutagenesis, ChIP-sequencing, Activation Assay, Luciferase, Generated