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Characterization of radioresistant epithelial stem cell heterogeneity in the damaged mouse intestine

ABSTRACT: The small intestine has a robust regenerative capacity, and various cell types serve as “cells-of-origin” in the epithelial regeneration process after injury. However, how much each population contributes to regeneration remains unclear. Using lineage tracing, we found that Lgr5-expressing cells’ derivatives contained radioresistant ISCs crucial for epithelial regeneration in the damaged intestine after radiation. Single-cell RNA sequencing analysis of Lgr5-derivatives identified primary source of epithelial regeneration in the irradiation damaged intetsine.

ORGANISM(S): Mus musculus

PROVIDER: GSE146783 | GEO | 2020/05/22

REPOSITORIES: GEO

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Project description:Background & Aims: Hierarchical organization of intestine relies on their stem cells by self-renew and producing committed progenitors. Although signals like Wnt are known to animate the continued renewal by maintaining intestinal stem cells (ISCs) activity, molecular mechanisms especially E3 ubiquitin ligases that modulate ISCs ‘stemness’ and supportive niche have not been well understood. Here, we investigated the role of Cullin 4B (Cul4b) in regulating ISC functions. Methods: We generated mice with intestinal epithelial-specific disruption of Cul4b (pVillin-cre; Cul4bfn/Y), inducible disruption of Cul4b (Lgr5-creERT2; Cul4bfn/Y, CAG-creERT2; Cul4bfn/Y) and their control (Cul4bfn/Y). Intestinal tissues were analyzed by histology, immunofluorescence, RNA sequencing and mass spectrum. Intestinal organoids deprived from mice with pVillin-Cre; Cul4bfn/Y, Lgr5-Cre; Cul4bfn/Y, Tg-Cul4b and their controls were used in assays to measure intestinal self-renewal, proliferation and differentiation. Wnt signaling and intestinal markers were analyzed by immunofluorescence and immunoblot assays. Differential proteins upon Cul4b ablation or Cul4b-interacting proteins were identified by mass spectrometry. Results: Cul4b specifically located at ISCs zone. Block of Cul4b impaired intestinal homeostasis maintenance by reduced self-renewal and proliferation. Transcriptome analysis revealed that Cul4b-null intestine lose ISC characterization and showed disturbed ISC niche. Mechanistically, reactivated Wnt pathway could recover intestinal dysfunction of Cul4b knockout mice. Analysis of differential total and ubiquitylated proteins uncovered the novel targeting substrate of Cullin-Ring ubiquitin ligase 4b (CRL4b), immunity-related GTPase family M member 1 (Irgm1) in intestine. Decreased Irgm1 rescued abnormally interferon signaling, overemphasized autophagy and downstream phosphate proteins in Cul4b knockout mice. Conclusion: We conclude that Cul4b is essential for ISC self-renewal and Paneth cell function by targeting Irgm1 and modulating Wnt signaling. Our results demonstrate that Cul4b is a novel ISC stemness and niche regulator.

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Characterization of radioresistant epithelial stem cell heterogeneity in the damaged mouse intestine

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