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. 2018 May;557(7704):242-246.
doi: 10.1038/s41586-018-0084-4. Epub 2018 May 2.

Subepithelial telocytes are an important source of Wnts that supports intestinal crypts

Michal Shoshkes-Carmel  1 , Yue J Wang  1 , Kirk J Wangensteen  1 , Beáta Tóth  2 , Ayano Kondo  1 , Efi E Massasa  2 , Shalev Itzkovitz  2 , Klaus H Kaestner  3
Affiliations

Subepithelial telocytes are an important source of Wnts that supports intestinal crypts

Michal Shoshkes-Carmel et al. Nature. 2018 May.

Erratum in

Abstract

Tissues that undergo rapid cellular turnover, such as the mammalian haematopoietic system or the intestinal epithelium, are dependent on stem and progenitor cells that proliferate to provide differentiated cells to maintain organismal health. Stem and progenitor cells, in turn, are thought to rely on signals and growth factors provided by local niche cells to support their function and self-renewal. Several cell types have been hypothesized to provide the signals required for the proliferation and differentiation of the intestinal stem cells in intestinal crypts1-6. Here we identify subepithelial telocytes as an important source of Wnt proteins, without which intestinal stem cells cannot proliferate and support epithelial renewal. Telocytes are large but rare mesenchymal cells that are marked by expression of FOXL1 and form a subepithelial plexus that extends from the stomach to the colon. While supporting the entire epithelium, FOXL1+ telocytes compartmentalize the production of Wnt ligands and inhibitors to enable localized pathway activation. Conditional genetic ablation of porcupine (Porcn), which is required for functional maturation of all Wnt proteins, in mouse FOXL1+ telocytes causes rapid cessation of Wnt signalling to intestinal crypts, followed by loss of proliferation of stem and transit amplifying cells and impaired epithelial renewal. Thus, FOXL1+ telocytes are an important source of niche signals to intestinal stem cells.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended Data Fig. 1
Extended Data Fig. 1. Fluorescence-activated cell sorting plots
(a–d) Representative FACS plots of mesenchymal cells isolated from Foxl1 Cre; Rosa-mTmG (c–d) as compared to control Rosa-mTmG (a–b) mice, showing sorting strategy. (a,c) Allophycocyanin+ (APC+) CD45+ and EpCAM+ labeled cells were gated out to exclude immune and epithelial cell contamination. (d) Foxl1+, GFP+ and Foxl1− Tomato+ cells were gated and sorted based on fluorescence activity as compare to the negative control (b). Experiments were repeated for at least three times with similar results.
Extended Data Fig. 2
Extended Data Fig. 2. Single Cell qPCR of Foxl1+ telocytes showing heterogeneity within Foxl1+ cell population
Hierarchical clustering of Foxl1+ single cells based on qPCR-based detection of 30 genes. Note that all Foxl1+ cells express Pdgfra and Wnt3a. The population is clustered into three main groups. Jaccard coefficient for the three clusters were: 0.83 (green), 0.69 (turquoise) and 0.79 (yellow), respectively, indicating underlying cluster stability. Values between 0.6 and 0.75 indicate that the cluster is measuring a pattern in the data. Clusters with stability values above about 0.85 are considered to be highly stable.
Extended Data 3
Extended Data 3. Derivation of Foxl1CreERT2;PorcnΔ mice
(a) Schema for the generation of Foxl1-CreERT2 mice using BAC recombineering. The coding sequence of exon 1 of Foxl1 was targeted by the sequence of CreERT2. FRT, flipase recognition target; Flp, flipase; LA, left homology arm; RA, right homology arm. (b–c) Tamoxifen Induction of Foxl1-CreERT;Rosa-mTmG drive expression of membrane-bound GFP to mesenchymal telocytes in the duodenum (b) and Colon (c). Immunofluorescece staining for GFP (green), EpCAM (red). (d) Schema for the generation of Foxl1CreERT2;PorcnΔ mice. Foxl1-CreERT2 mice were crossed to mice carrying loxP sites flanking exons 3–7 of the X-linked porcupine homolog (Porcn) gene. (e) Foxl1-CreERT2; Rosa-mTmG (Control, n=5 biologically independent animals) and Foxl1-CreERT2;PorcnΔ (PorcnΔ, n=8 biologically independent animals) male mice were tamoxifen treated for three consecutive days to induce Cre expression and weighed every day. The slope in weight loss was significantly different in PorcnΔ mice as compared to control mice (*P=0.0107, two-tailed linear regression analysis)
Figure 1
Figure 1. Foxl1+ cells are telocytes and co-express PDGFRα
(a) Schema for labeling Foxl1+ cells with GFP. (b) Foxl1-Cre-driven GFP is restricted to pericryptal mesenchymal telocytes. Immunofluorescence staining for Foxl1 (white, see asterisks), GFP (green), EpCAM (red) on cleared mouse whole duodenum. (c,d) Immunofluorescence for GFP (green) and PDGFRα (red) in the duodenum (c) and colon (d). (e–g) GFP immuno-electron microscopy of Foxl1-Cre;Rosa-mTmG duodenal crypt (transverse section). (f) Inset showing the telocyte nucleus and its extension, the telopode. n; nucleus, Tp; telopode. (g) Inset of contact between two telopodes. (h) Confocal imaging of cleared mouse whole small intestine. PDGFRα (green) and EpCAM (red). Experiments were repeated for at least three times with similar results. Scale bars represent 10μm.
Figure 2
Figure 2. Telocytes express key signaling molecules
RNAseq analysis of Foxl1+ cells (n=3 biologically independent animals) compared to Foxl1− mesenchymal cells (n=3 biologically independent animals), Lgr5+ stem cells (n=2 biologically independent animals), and differentiated enterocytes (n=2 biologically independent animals). (a) Heatmap showing hierarchical clustering. Only genes with a fold-change > 10 in pairwise comparisons and an FDR < 2% were used for the analysis. Values are normalized z-scores, plus or minus standard deviation. (b–g) Selected markers showing differential expression: (b) Foxl1, Pdgfra, Cd34 (telocyte markers), Lgr5 and Slc5a1, (c) Wnt pathway, (d) Bmp pathway, (e) Shh pathway, (f) Growth factors, (g) Tgfβ pathway. Data are shown as FPKM. Error bars indicate SEM.
Figure 3
Figure 3. Telocytes compartmentalize signaling molecule mRNAs for localized signaling
(a–d) Cryosections of mouse duodenum stained for PDGFRα (blue), DNA (gray), and hybridized with single-molecule FISH probes as indicated. Insets ‘1,2,3’ represent large magnification of selected areas. (e) For mRNA quantification, fluorescence signals were counted per defined area along the crypt-villus axis. (f–k) mRNA molecules per cubic micron along the crypt-villus axis. Experiments were repeated for at least three times with similar results. Scale bars represent 10μm. Black bars represent the mean values. ***P < 0.001, *P < 0.1 ordinary one-way ANOVA multiple comparison with nonparametric Kruskal-Wallis test.
Figure 4
Figure 4. The intestinal epithelium depends on Wnts secreted from Foxl1+ telocytes
(a–c) Control and PorcnΔ mutant duodenum, one and three days post tamoxifen injection. Experiments were repeated for at least three times with similar results. (d,e) Duodenal crypt depth (Control n=3 biologically independent animals, PorcnΔ 24hr n=3 biologically independent animals) and villus length (Control n=9 biologically independent animals, PorcnΔ 24hr n=3 biologically independent animals, PorcnΔ 72hr n=7 biologically independent animals,, ***P < 0.001, unpaired two-tailed t test) (f–h) Control and PorcnΔ mutant colon, one and three days post tamoxifen injection. Experiments were repeated for at least three times with similar results. (l) Colonic crypt depth (Control n=7 biologically independent animals, PorcnΔ 72hr n=3 biologically independent animals, PorcnΔ 72hr n=7 biologically independent animals, ***P < 0.001, unpaired two-tailed t-test). (j–l,n–p) EdU incorporation (red) in the epithelium (EpCAM, green) of duodenum and colon. Experiments were repeated for at least three times with similar results. (m) Proliferating cells per duodenal crypt. (n=3 biologically independent animals per group ***P < 0.001, unpaired two-tailed t test). (r) Proliferating cells per colonic crypt. (n=3 biologically independent animals per group ***P < 0.001, unpaired two tailed t-test). Center line indicate mean and Error bars indicate SD, Scale bars 100μm.
Figure 5
Figure 5. Foxl1+ telocytes provide essential Wnt ligands to the intestinal stem cell compartment
(a,b) Wnt pathway activation analyzed by immunohistochemistry for β catenin (brown). Insets a′ and b′ at high magnification. (c–j) Immunofluorescence staining of the Wnt targets CyclinD1 and Sox9. (k–n) Stem cell markers Olfm4 (in situ hybridization) and CD44 (immunohistochemistry) one day post induction in PorcnΔ duodenum. Insets k′, l′, m′, n′ represent high magnification. (o,p) Expression of Lgr5 and Rspo3 as detected by single molecule RNA-FISH three day post induction in PorcnΔ duodenum. * denotes a staining artifact. (q,r) Immunofluorescence for EpCAM and PDGRFα in duodenum of control and PorcnΔ mice. Experiments were repeated for at least three times with similar results. Scale bars 100 μm (a–n) and 25 μm in insets
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