The other is a slower cycling or quiescent cell population that is thought to reside above CBCs and Paneth cells and has been termed the +4 ISCs because of their predominant location at approximately four cells from the base of the crypt (33, 46). Recent evidence suggests that the +4 or quiescent ISC population may be marked by Bmi1 (54), Hopx selleck bio (61), doublecortin and CaM kinase-like-1 (DCAMKL-1) (39), or EEC markers (57). Furthermore, a bidirectional lineage relationship between active CBCs and +4 ISCs has been recently demonstrated (61, 64). In this study, we hypothesized that, following crypt and ISC ablation after high-dose radiation, crypt regeneration would involve expansion, hyperproliferation, and altered molecular phenotype of Sox9-EGFP Low cells, which correspond to active ISCs.

To test our hypothesis, we characterized changes in Sox9-EGFP cell populations after irradiation using histology and flow cytometry. We also assessed the ability of Sox9-EGFP-expressing cells isolated during irradiation-induced regeneration to form organoids in vitro (21, 55) and used microarray to define gene expression changes exhibited by each Sox9-EGFP cell population during crypt regeneration. Our findings support a major role of Sox9-EGFP Low ISCs in crypt regeneration but also provide evidence for an additional ISC population contained within Sox9-EGFP High cells that is activated to proliferate during irradiation-induced crypt regeneration. MATERIALS AND METHODS Animals Mice expressing a BAC transgene with ~226.

5 kb of Sox9 genomic regulatory region driving EGFP expression were established and maintained at the University of North Carolina (Chapel Hill, NC) as previously described (17, 21). Sox9-EGFP mice are on the outbred CD-1 strain and were maintained as heterozygotes by breeding with wild-type CD-1 strain mice. Genotyping was performed as in Refs. 17 and 21. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina. Abdominal Irradiation Mice were given a single dose of 14 Gy irradiation by using an XRad 320 (Precision X-Ray, East Haven, CT) (Filter: 2 mm Al; 47 cm; 320 kV/s, 10 mA; 2.8 Gy/min). All radiation experiments were performed under isoflurane anesthesia, and mice were placed in the radiator so that only the abdomen lay in the radiated zone. Body weight was recorded every day.

Typically, mice lost up to 25% body weight until day 7 postirradiation when body weight started to increase toward normal. Pilot studies indicated that CD-1 strain mice survived and began to gain weight even after losing the maximum 15% body weight typically used to warrant Batimastat euthanasia. Our approved IACUC protocol therefore permitted an exception to euthanize mice only if they lost more than 25% body weight.