SOX9 maintains reserve stem cells and preserves radioresistance in mouse small intestine

Gastroenterology. 2015 Nov;149(6):1553-1563.e10. doi: 10.1053/j.gastro.2015.07.004. Epub 2015 Jul 11.


Background & aims: Reserve intestinal stem cells (rISCs) are quiescent/slowly cycling under homeostatic conditions, allowing for their identification with label-retention assays. rISCs mediate epithelial regeneration after tissue damage by converting to actively proliferating stem cells (aISCs) that self renew and demonstrate multipotency, which are defining properties of stem cells. Little is known about the genetic mechanisms that regulate the production and maintenance of rISCs. High expression levels of the transcription factor Sox9 (Sox9(high)) are associated with rISCs. This study investigates the role of SOX9 in regulating the rISC state.

Methods: We used fluorescence-activated cell sorting to isolate cells defined as aISCs (Lgr5(high)) and rISCs (Sox9(high)) from Lgr5(EGFP) and Sox9(EGFP) reporter mice. Expression of additional markers associated with active and reserve ISCs were assessed in Lgr5(high) and Sox9(high) populations by single-cell gene expression analyses. We used label-retention assays to identify whether Sox9(high) cells were label-retatining cells (LRCs). Lineage-tracing experiments were performed in Sox9-CreERT2 mice to measure the stem cell capacities and radioresistance of Sox9-expressing cells. Conditional SOX9 knockout mice and inducible-conditional SOX9 knockout mice were used to determine whether SOX9 was required to maintain LRCs and rISC function.

Results: Lgr5(high) and a subset of crypt-based Sox9(high) cells co-express markers of aISC and rISC (Lgr5, Bmi1, Lrig1, and Hopx). LRCs express high levels of Sox9 and are lost in SOX9-knockout mice. SOX9 is required for epithelial regeneration after high-dose irradiation. Crypts from SOX9-knockout mice have increased sensitivity to radiation, compared with control mice, which could not be attributed to impaired cell-cycle arrest or DNA repair.

Conclusions: SOX9 limits proliferation in LRCs and imparts radiation resistance to rISCs in mice.

Keywords: Gene Expression; Intestinal Epithelium; Irradiation Injury; Radioresistant Stem Cells.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Proliferation / physiology
  • Enterocytes / metabolism*
  • Enterocytes / radiation effects
  • Gene Expression
  • Green Fluorescent Proteins / metabolism
  • Intestinal Mucosa / cytology*
  • Intestinal Mucosa / metabolism
  • Intestinal Mucosa / radiation effects
  • Intestine, Small / metabolism
  • Intestine, Small / radiation effects*
  • Mice
  • Mice, Knockout
  • Polycomb Repressive Complex 1 / metabolism
  • Radiation Tolerance / genetics
  • Radiation Tolerance / physiology*
  • Receptors, G-Protein-Coupled / metabolism
  • Regeneration / physiology
  • SOX9 Transcription Factor / genetics
  • SOX9 Transcription Factor / metabolism*
  • Stem Cells / cytology
  • Stem Cells / metabolism*
  • Stem Cells / radiation effects


  • BMI1 protein, human
  • Lgr5 protein, mouse
  • Receptors, G-Protein-Coupled
  • SOX9 Transcription Factor
  • Sox9 protein, mouse
  • Green Fluorescent Proteins
  • Polycomb Repressive Complex 1