A stromal lineage maintains crypt structure and villus homeostasis in the intestinal stem cell niche

Abstract

Background: The nutrient-absorbing villi of small intestines are renewed and repaired by intestinal stem cells (ISCs), which reside in a well-organized crypt structure. Genetic studies have shown that Wnt molecules secreted by telocytes, Gli1⁺ stromal cells, and epithelial cells are required for ISC proliferation and villus homeostasis. Intestinal stromal cells are heterogeneous and single-cell profiling has divided them into telocytes/subepithelial myofibroblasts, myocytes, pericytes, trophocytes, and Pdgfra^low stromal cells. Yet, the niche function of these stromal populations remains incompletely understood.

Results: We show here that a Twist2 stromal lineage, which constitutes the Pdgfra^low stromal cell and trophocyte subpopulations, maintains the crypt structure to provide an inflammation-restricting niche for regenerating ISCs. Ablating Twist2 lineage cells or deletion of one Wntless allele in these cells disturbs the crypt structure and impairs villus homeostasis. Upon radiation, Wntless haplo-deficiency caused decreased production of anti-microbial peptides and increased inflammation, leading to defective ISC proliferation and crypt regeneration, which were partially rescued by eradication of commensal bacteria. In addition, we show that Wnts secreted by Acta2⁺ subpopulations also play a role in crypt regeneration but not homeostasis.

Conclusions: These findings suggest that ISCs may require different niches for villus homeostasis and regeneration and that the Twist2 lineage cells may help to maintain a microbe-restricted environment to allow ISC-mediated crypt regeneration.

Keywords: ISC; Inflammation; Mesenchymal; Niche; Paneth cell; Wntless.

Fig. 1

Lineage tracing and scRNA-seq analysis of Twist2 lineage cells in the intestinal villi. a Detection of stromal lineages labeled by Twist2, Prrx1, or Nestin, and Acta2⁺ myofibroblasts in the small intestinal villi of adult mice. Right panel: the percentage of Twist2, Prrx1, and Nestin lineage cells and Acta2 lineage myofibroblasts in non-epithelial (EpCAM⁺) cells. Bar: 50 μm. The calculation was done by counting Tomato⁺ cells on six views per mouse and divided by the number of non-epithelial DAPI⁺ nuclei, N = 6. b Single-cell transcriptome analysis of small intestinal cells. A scRNA-seq dataset (PMID 32084389) was used to analyze the expression of various stromal/myoblast cell markers

Fig. 2

Depletion of Twist2 lineage cells impaired villus homeostasis. a Depletion of the Twist2 or Acta2 lineage, but not the Prrx1 or Nestin lineage, led to shortening of the GI tracts. Right panel: quantitation data of the intestine length. N = 4. See additional file: Fig. S3e for details. b H/E, Ki67, goblet, lysozyme, and GFP staining of normal and Twist2 cell-depleted mouse intestinal villi. Also, see Fig. S3g for control. Bar: 50 μm. Bottom panels: quantitation data. **P < 0.01 and ***P < 0.001. ns: not significant. N = 5. Lgr5-GFP: Lgr5-EGFP-IRES-CreERT2

Fig. 3

Intestinal Twist2 cell-secreted Wnts were critical for villus homeostasis and crypt structure maintenance. a Expression of Wnt molecules in Twist2 intestinal stromal cells. Intestinal Tomato⁺ stromal cells were sorted from Twist2-Cre; Tomato mice and collected for RNA isolation and qPCR analysis. The Value of Wnt1 was set at 1.0. N = 3. b Quantitative PCR results showed that stromal cells (Tomato⁺) of the small intestine of Twist2-Cre;Tomato;Wls^+/f mice showed a decrease in the mRNA levels of Wls compared to control mice. N = 4. c Representative immunofluorescent staining results for β-Catenin in the intestinal villi of control and Twist2-Cre;Wls^+/f mice. Bar: 50 μm. The intestine sections were stained with anti-β-Catenin antibodies and DAPI. β-Catenin immunofluorescent staining results without DAPI is shown in additional file: Fig. S6. d H/E, Ki67, goblet, Paneth cells, and GFP staining of intestinal villi of control and Twist2-Cre;Wls.^+/f mice. Bar: 50 μm. Bottom panels: quantitation data. *P < 0.05, **P < 0.01, and ***P < 0.001. N = 4 (% of Ki67 + cells/crypt and # of Paneth cells/crypt). N = 6 (# of Goblet cells/villus). Others N = 5

Fig. 4

Twist2 cell-secreted Wnts were critical for villus and crypt development. a Detection of Twist2 lineage cells in E14.5, E16.5, and p12 Twist2-Cre;Tomato mouse intestines. Bar: 50 μm. b Quantitative PCR results of the intestinal stromal cells (Tomato⁺) of E16.5 control, Twist2-Cre;Wls^f/f, and Twist2-Cre;Wls^+/f embryos. N = 4. c Representative immunohistochemical staining results for β-Catenin in the intestinal villi of E16.5 control, Twist2-Cre;Wls^f/f, and Twist2-Cre;Wls^+/f embryos. Bar: 50 μm. Brown color indicated β-Catenin signals. d H/E, Ki67, and goblet staining of intestinal villi of E16.5 control, Twist2-Cre;Wls^f/f, and Twist2-Cre;Wls^+/f embryos. Bar: 50 μm. Bottom panels: quantitation data. ***P < 0.001. N = 8 or N = 7 (Ki67). e Quantitative PCR results of the intestinal stromal cells (Tomato⁺) of P12 control and Twist2-Cre;Wls^+/f pups. N = 4. Brown color indicated β-Catenin signals. f Representative immunohistochemical staining results for β-Catenin in the intestinal villi of P12 control and Twist2-Cre;Wls^+/f pups. Bar: 50 μm. g H/E, Ki67, goblet, and Paneth cell staining of intestinal villi of p12 control and Twist2-Cre; Wls.^f/f embryos. Bar: 50 μm. Bottom panels: quantitation data. *P < 0.1, **P < 0.01, and ***P < 0.001. N = 7

Fig. 5

Intestinal Twist2 cell-secreted Wnts were critical for crypt regeneration. a H/E, Ki67, goblet, and Paneth cell staining of intestinal villi of control and Twist2-Cre;Wls^+/f mice 4 or 7 days after IR (6.5 Gy). Bar: 50 μm. Right panels: quantitation data. *P < 0.05, **P < 0.01, and ***P < 0.001. N = 4. b Acta2-Cre; Wls^f/f mice showed a defect in crypt regeneration 4 days after IR. H/E, Ki67, and goblet cell staining of intestinal villi of control and Acta2-Cre; Wls.^f/f mice 4 days after IR (6.5 Gy). Bar: 50 μm. Right panels: quantitation data. *P < 0.05, **P < 0.01, and***P < 0.001. N = 5

Fig. 6

Crypt regeneration defects in Twist2-Cre; Wls^+/f mice were rescued by antibiotics treatment. a Decreases in the number of Paneth cells and expression of AMPs in regenerating villi of Twist2-Cre; Wls^+/f mice. Bar: 50 μm. b qPCR analysis showed reduced expression of Crypt4 in the villi of villi of Twist2-Cre;Wls^+/f mice. The basal levels in Twist2-Cre; Wls^+/f mouse intestines were set at 1.0. ***P < 0.001. N = 4. c IR-induced production of inflammatory cytokines in the intestine was enhanced in Twist2-Cre; Wls^+/f mice, which was rescued by antibiotic treatment. The basal levels of cytokines in normal mouse intestines were set at 1.0. *P < 0.05, **P < 0.01, and ***P < 0.001. N = 3. d Crypt regeneration defects in Twist2-Cre; Wls.^+/f mice were rescued by antibiotics treatment. Bar: 50 μm. Bottom panels: quantitation data. *P < 0.05, **P < 0.01, and ***P < 0.001. N = 5