title: Activation of two distinct Sox9-EGFP-expressing intestinal stem cell populations during crypt regeneration after irradiation.
abstract: Recent identification of intestinal epithelial stem cell (ISC) markers and development of ISC reporter mice permit visualization and isolation of regenerating ISCs after radiation to define their functional and molecular phenotypes. Previous studies in uninjured intestine of Sox9-EGFP reporter mice demonstrate that ISCs express low levels of Sox9-EGFP (Sox9-EGFP Low), whereas enteroendocrine cells (EEC) express high levels of Sox9-EGFP (Sox9-EGFP High). We hypothesized that Sox9-EGFP Low ISCs would expand after radiation, exhibit enhanced proliferative capacities, and adopt a distinct gene expression profile associated with rapid proliferation. Sox9-EGFP mice were given 14 Gy abdominal radiation and studied between days 3 and 9 postradiation. Radiation-induced changes in number, growth, and transcriptome of the different Sox9-EGFP cell populations were determined by histology, flow cytometry, in vitro culture assays, and microarray. Microarray confirmed that nonirradiated Sox9-EGFP Low cells are enriched for Lgr5 mRNA and mRNAs enriched in Lgr5-ISCs and identified additional putative ISC markers. Sox9-EGFP High cells were enriched for EEC markers, as well as Bmi1 and Hopx, which are putative markers of quiescent ISCs. Irradiation caused complete crypt loss, followed by expansion and hyperproliferation of Sox9-EGFP Low cells. From nonirradiated intestine, only Sox9-EGFP Low cells exhibited ISC characteristics of forming organoids in culture, whereas during regeneration both Sox9-EGFP Low and High cells formed organoids. Microarray demonstrated that regenerating Sox9-EGFP High cells exhibited transcriptomic changes linked to p53-signaling and ISC-like functions including DNA repair and reduced oxidative metabolism. These findings support a model in which Sox9-EGFP Low cells represent active ISCs, Sox9-EGFP High cells contain radiation-activatable cells with ISC characteristics, and both participate in crypt regeneration.
authors.affiliation: Department of Cellular and Molecular Physiology, University of North Carolina, Chapel Hill, NC 27599-7545, USA.
authors.forename: Laurianne
authors.initials: L
authors.lastname: Van Landeghem
authors.forename: M Agostina
authors.initials: MA
authors.lastname: Santoro
authors.forename: Adrienne E
authors.initials: AE
authors.lastname: Krebs
authors.forename: Amanda T
authors.initials: AT
authors.lastname: Mah
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Dehmer
authors.forename: Adam D
authors.initials: AD
authors.lastname: Gracz
authors.forename: Brooks P
authors.initials: BP
authors.lastname: Scull
authors.forename: Kirk
authors.initials: K
authors.lastname: McNaughton
authors.forename: Scott T
authors.initials: ST
authors.lastname: Magness
authors.forename: P Kay
authors.initials: PK
authors.lastname: Lund
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000528:radiation effects; D002478:Cells, Cultured; D015870:Gene Expression / Q000528:radiation effects; D049452:Green Fluorescent Proteins / Q000096:biosynthesis*; D018398:Homeodomain Proteins / Q000096:biosynthesis; D007583:Jejunum / Q000378:metabolism / Q000528:radiation effects*; D051379:Mice; D008822:Mice, Transgenic; D009687:Nuclear Proteins / Q000096:biosynthesis; D063150:Polycomb Repressive Complex 1; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis; D012038:Regeneration / Q000528:radiation effects; D012097:Repressor Proteins / Q000096:biosynthesis; D055758:SOX9 Transcription Factor / Q000096:biosynthesis*; D013234:Stem Cells / Q000528:radiation effects*
chemical_list: C489421:Bmi1 protein, mouse; C473013:Hod protein, mouse; D018398:Homeodomain Proteins; C493597:Lgr5 protein, mouse; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D043562:Receptors, G-Protein-Coupled; D012097:Repressor Proteins; D055758:SOX9 Transcription Factor; C527933:Sox9 protein, mouse; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D063150:Polycomb Repressive Complex 1