Fig 1: CXCL12 partially rescues impaired regeneration of intestinal mucosa in EC‐Foxc‐DKO mice after I/RIn CXCL12 rescue experiments, mice were treated with 50 μg/kg CXCL12 in PBS by retro‐orbital injection 30 min before ischemia. Mice treated with PBS were used as control. ARepresentative images of H&E staining show the rescue effects of CXCL12 in intestinal mucosa in control and EC‐Foxc‐DKO mice 24 h after I/R. Red numbers indicate Chiu scores. Scale bars = 100 μm.BQuantification of Chiu Scores for mouse intestines 24 h after I/R based on H&E staining as shown in Fig 10A. Data are box‐and‐whisker plots, Mann–Whitney U test, each symbol represents one mouse, N = 6 ~ 11, *P < 0.05.CRepresentative images of crypts immunostained with OLFM4 and β‐catenin in PBS‐ and CXCL12‐treated EC‐Foxc‐DKO mice 24 h after I/R. The total protein signal of β‐catenin is upregulated in CXCL12‐treated crypts compared with the PBS‐treated group. The accumulation of β‐catenin in the nuclei of ISCs (dotted circles) was found in CXCL12‐treated mice but inhibited in PBS‐treated mice. Paraffin sections (4 μm), scale bars = 20 μm.D, E(D) Quantification of relative fluorescent intensity (FI) of β‐catenin immunostaining within ISC and (E) quantification of the number of OLFM4+ ISCs per crypt were performed based on Fig 10C. Data are box‐and‐whisker plots, Mann–Whitney U test, each symbol represents one mouse, N = 5 ~ 7, *P < 0.05.F, G(F) Representative immunostaining images of CCND1 and (G) Quantification of CCND1+ epithelial cells per crypt after I/R at 24 h. Scale bars = 100 μm. Data are box‐and‐whisker plots, Mann–Whitney U test, each symbol represents one mouse, N = 7 ~ 8, ***P < 0.001.HRepresentative confocal images of CD31 immunostaining of distal jejuna in PBS‐ and CXCL12‐ treated EC‐Foxc‐DKO mice after I/R at 24 h. Paraffin sections (15 μm), scale bars = 100 μm. L represents the lacteal length measured in Fig 10J.I, J(I) Quantification of CD31+ vessel density (% = total CD31+ vessel area/total intestinal tissue area × 100%) and (J) quantification of lacteal length were performed based on Fig 10H. Data are box‐and‐whisker plots, Mann–Whitney U test, each symbol represents one mouse, N = 4 ~ 6, **P < 0.01.KRelative mRNA expression of Rspo3 in sorted intestinal LECs from PBS/CXCL12‐treated EC‐Foxc‐DKO mice at I/R‐18.5 h. Data are box‐and‐whisker plots, Mann–Whitney U test, each symbol represents one mouse, N = 5 ~ 6, n.s., not significant. Data information: The box‐and‐whisker plots in (B), (D), (E), (G) and (I–K) display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively), as well as minimum/maximum values (whiskers blow/above the box) of the data sets. Source data are available online for this figure.
Fig 2: ScRNA-seq analysis on the small intestines from control and EC-Foxc-DKO mice 18.5 h after I/R Pie chart showing the percentage of each cell cluster identified in Fig 6A of total cell population.Sub-clustering performed on the cluster 20 (Telocytes/Trophocytes).Dot plot showing relative expression of different genes identified by scRNA-seq were decreased in crypt cell cluster in EC-Foxc-DKO mice compared with control mice at I/R-18.5 h. Fill colors represent normalized mean expression levels and circle sizes represent the within-cluster frequency of positive gene detection. Lgr5, Olfm4, Ascl2, Sox9, Cd24a, Smoc2 and Aldh1b1 are ISC markers. Mik67 is a proliferative marker. Ascl2, Sox9 and Axin2 are Wnt/ß-catenin target genes.Schematic drawing of the mechanism by which endothelial FOXC1 and FOXC2 promote mouse intestinal regeneration after I/R injury. The intestinal mucosa is damaged after I/R injury. FOXC (both FOXC1 and FOXC2) regulate the expression of Rspo3/Cxcl12 through binding to their regulatory elements in LECs/BECs of the lymphatic/blood vessels near the crypts, respectively. RSPO3 secreted by LECs (blue arrow) is an agonist of the canonical Wnt/ß-catenin signaling pathway and promotes the intestinal epithelial regeneration and repair. RSPO3 derived from LECs also promotes angiogenesis and lymphangiogenesis (dashed arrows). BEC-derived CXCL12 (pink arrow) not only regulates angiogenesis (arrow) but also stimulates CXCR4 on LECs to enhance lymphangiogenesis (dashed arrow). L, lymphatic vessel; B, blood vessel; ISC, intestinal stem cell; LEC, lymphatic endothelial cell; BEC, blood endothelial cell. Source data are available online for this figure.
Fig 3: FOXC1 and FOXC2 regulate the expression of RSPO3 in LECs and CXCL12 in BECs through binding to their regulatory elements A–D(A and B) In silico identification of putative FOX-binding sites in the RSPO3 and CXCL12 loci. Putative FOX-binding sites in regions of the human RSPO3 (A) and CXCL12 (B) loci as viewed on the UCSC genome browser (https://genome.ucsc.edu; Kent et al, 2002). Vertical lines on the “FOX sites” and “FOXC sites” tracks indicate putative binding sites corresponding to the FOX “RYMAAYA” consensus sequence and the FOXC “RYACACA” consensus sequence (Chen et al, 2019) predicted using the Hypergeometric Optimization of Motif EnRichment (HOMER) suite of tools (Heinz et al, 2010). Red boxes indicate evolutionary conserved regions (ECRs) containing FOX-binding sites between human and mouse genomes that are conserved and aligned as identified using the ECR browser tool (ecrbrowser.docde.org). Green boxes indicate promoter regions in CXCL12 containing FOX-binding sites according to the prediction of JASPAR. The red arrows indicate the site of transcription initiation for RSPO3 or CXCL12. (C and D) FOXC1 and FOXC2 co-occupy the ECRs of RSPO3 in HDLECs (C), as well as the ECRs and promoters of CXCL12 in HUVECs (D). Quantitative-ChIP assay was performed using rabbit (R) anti-FOXC1 and mouse (M) anti-FOXC2 antibodies to analyze the recruitment of FOXC on promoters and/or ECRs in HDLECs and HUVECs respectively. Values were quantified against IgG controls. Data are mean ± SEM, paired t-test, each symbol represents data collected from one experiment, N = 4, *P < 0.05, **P < 0.01, n.s., not significant. Source data are available online for this figure.
Fig 4: RSPO3 partially rescues impaired regeneration of intestinal mucosa in EC‐Foxc‐DKO and LEC‐Foxc‐DKO mice after I/RIn RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100 μl PBS by retro‐orbital injection 30 min before ischemia. PBS‐treated mice were used as vehicle control. A–J(A and I) Representative images of H&E staining show the rescue effects of RSPO3 in intestinal mucosa in EC‐Foxc‐DKO (A) and LEC‐Foxc‐DKO (I) mice, as well as their control mice 24 h after I/R. Red numbers indicate the Chiu scores. Scale bars = 100 μm. (B and J) Quantification of Chiu Score for the intestines at I/R‐24 h is based on H&E staining as shown in Fig 9A and I. Data are box‐and‐whisker plots, Mann–Whitney U test, each symbol represents one mouse, N = 6 ~ 13, **P < 0.01. (C) Representative images of crypts immunostained with OLFM4 and β‐catenin in PBS/RSPO3 treated EC‐Foxc‐DKO mice 24 h after I/R. The accumulation of β‐catenin in the nuclei of ISCs (dotted circles) was found in RSPO3‐rescued mice. Paraffin sections (4 μm), scale bars = 20 μm. (D) Quantification of relative fluorescent intensity (FI) of β‐catenin immunostaining within ISC and (E) quantification of the number of OLFM4+ ISCs were performed based on Fig 9C. Data are box‐and‐whisker plots, Mann–Whitney U test, each symbol represents one mouse, N = 4 ~ 6, *P < 0.05. (F) Representative images of immunostaining of CCND1 in intestines in PBS/RSPO3 treated EC‐Foxc‐DKO mice at I/R‐24 h. Scale bars = 100 μm. (G) Quantification of the number of CCND1+ epithelial cells per crypt at I/R‐24 h is based on Fig 9F. Data are box‐and‐whisker plots, Mann–Whitney U test, each symbol represents one mouse, N = 7, **P < 0.01. (H) Relative mRNA expression of Rspo3 in sorted LECs and Cxcl12 in sorted BECs from intestines of PBS/RSPO3 treated EC‐Foxc‐DKO mice at I/R‐18.5 h. Data are box‐and‐whisker plots, Mann–Whitney U test, each symbol represents one mouse, N = 4 ~ 6, *P < 0.05.KRepresentative images of CD31/LYVE1 immunostaining in the intestines of PBS/RSPO3 treated LEC‐Foxc‐DKO mice at I/R‐24 h. Scale bars = 100 μm.L, MQuantification of the vessel density (L) (= vessel area/total intestinal tissue area × 100%) for the blood (B) and/or lymphatic (L) vessels (markers listed below the graph were used to identify B and L), as well as the measurement of lacteal length (M) was performed based on Fig 9K. The Data are box‐and‐whisker plots, Mann–Whitney U test, each symbol represents one mouse, N = 7, **P < 0.01, n.s., not significant. Data information: The box‐and‐whisker plots in (B), (D), (E), (G), (H), (J), (L) and (M) display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively), as well as minimum/maximum values (whiskers blow/above the box) of the data sets. Source data are available online for this figure.
Fig 5: Intestinal Rspo3 expression in control and EC‐Foxc‐DKO mice after I/R identified by single‐cell RNA sequencing Visualization of unsupervised clustering of 22 distinct clusters by UMAP from the distal jejunum of both control and EC‐Foxc‐DKO mice after I/R at 18.5 h.UMAP visualization of Rspo3 expression in three different cell clusters identified in Fig 6A.Dot plot showing relative expression of Rspo3 in five cell clusters identified by scRNA‐seq. Fill colors represent normalized mean expression levels and circle sizes represent the within‐cluster frequency of positive gene detection.Violin plots of the Rspo3 expression in trophocytes and LECs in the intestine at I/R‐18.5 h. Mann–Whitney U test, each symbol represents one cell; N = 12 and 12 in control and EC‐Foxc‐DKO trophocytes, respectively; N = 16 and 5 in control and EC‐Foxc‐DKO LECs.Validation study by qPCR for the detection of relative mRNA expression of Rspo3 in the sorted intestinal LECs at I/R‐18.5 h. Data are box‐and‐whisker plots, Mann–Whitney U test, each symbol represents one mouse, N = 5 ~ 6, **P < 0.01. The box‐and‐whisker plots display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively), as well as minimum/maximum values (whiskers blow/above the box) of the data sets. Source data are available online for this figure.
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