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Proc Natl Acad Sci U S A. Jan 4, 2005; 102(1): 99–104.
Published online Dec 22, 2004. doi:  10.1073/pnas.0405979102
PMCID: PMC544052
Cell Biology

Activated macrophages are an adaptive element of the colonic epithelial progenitor niche necessary for regenerative responses to injury

Abstract

We have identified cellular and molecular features of the stem cell niche required for marked amplification of mouse colonic epithelial progenitors (ColEPs) that occurs in response to wounding of the epithelium with dextran sodium sulfate. This regenerative response in areas adjacent to breaches in the epithelial barrier depends on the gut microbiota because ColEP proliferation is markedly diminished in germ-free animals. Analysis of conventionally raised C57BL/6 (B6) knockout mice lacking the Toll-like receptor signal transduction pathway component Myd88 and wild-type animals transplanted with Myd88-/- bone marrow, revealed that Myd88-mediated signaling through mesenchymal cells is also required for the ColEP response. Studies of B6 Csf1op/op (lacking macrophages) mice, Rag1-/- mice, and wild-type mice treated with neutrophil-specific Gr1 mAbs, disclosed that macrophages but not lymphocytes or neutrophils are necessary. GeneChip analysis of laser-capture-microdissected mesenchymal cells coupled with immunohistochemical and electron microscopic studies showed that, during the regenerative response, macrophages in the pericryptal stem cell niche express genes associated with their activation and extend processes to directly contact ColEPs near the crypt base. GeneChip analysis also identified a number of potential molecular mediators of regeneration expressed in the pericryptal progenitor niche, including secreted factors that stimulate epithelial proliferation and proteins involved in extracellular matrix and basement membrane function, stability, and growth factor binding. Together, these studies indicate that the colonic epithelial progenitor niche is a dynamic structure in which macrophages function as mobile “cellular transceivers” that coordinate inputs from luminal microbes and injured epithelium and transmit regenerative signals to neighboring ColEPs.

Keywords: laser capture microdissection, epithelial–mesenchymal cross-talk, macrophage activation, stem cell, stem cell niche

The epithelium that lines the inner surface of the human intestine can be disrupted by variety of environmental factors ranging from pathogenic bacteria to drugs and irradiation. Injury may lead to circumscribed loss of the epithelium, with formation of superficial erosions or more deeply penetrating ulcers. Repairing these lesions requires rapid recognition and response by the host because the intestinal lumen contains an abundant society of indigenous microbes (microbiota) that can cause opportunistic infection if the epithelial barrier is breached (1). A key initial phase of epithelial regeneration involves the expansion of epithelial progenitors in areas immediately adjacent to areas of damage. Delineating the cellular and molecular mechanisms that modulate this response to wounding provides an opportunity to identify cellular and molecular components that normally regulate the proliferative activities of multipotential and oligopotential epithelial lineage progenitors.

Colonic epithelial progenitors (ColEPs) reside in the lower half of mucosal invaginations known as crypts of Lieberkühn (2). ColEPs continuously give rise to three cell types: absorptive enterocytes plus two secretory cell lineages (goblet and enteroendocrine). Differentiating and differentiated cells migrate upwards from the crypt base to a surface epithelial cuff that surrounds the crypt orifice, where they undergo apoptosis and/or exfoliation (3). The migration of goblet cells is less uniform compared with the two other epithelial lineages, with the result that the lower half of crypts normally contains equal-sized, comingled populations of differentiated goblet cells and ColEPs.

Studies in many systems indicate the formation, proper functioning, and survival of stem cells is strongly influenced by their local microenvironment (niche; e.g., refs. 4 and 5). In the intestine, several mesenchymal constituents form structural components of the ColEP niche. Subepithelial myofibroblasts are a morphologic hybrid of smooth muscle cells and fibroblasts (6). Like their overlying epithelial cells, subepithelial myofibroblasts continuously migrate upward from the crypt base (7), placing them in strategic position to establish and maintain instructive communications with stem cells and their descendants. A submucosal plexus of enteric neurons communicates with a subset of crypt epithelial progenitors: their dendritic projections contact ColEPs, and there is evidence that they act as a key cellular intermediate in certain growth factor-signaling pathways (e.g., those involving glucagon-like peptide 2; see ref. 8). The microvasculature underlying the crypt base also effects ColEP survival. For example, radiation treatment triggers endothelial apoptosis, leading to ColEP death (9).

Multiple signaling pathways are recognized as contributing to the regulation of epithelial stem cell homeostasis in the adult mouse intestine. For example, Wnt signaling is a key regulator of proliferative status (10). Overexpression of Dickkopf-1, a secreted Wnt antagonist, dramatically inhibits proliferation and results in involution of crypts in adult mice (11, 12). This phenotype is similar to the loss of proliferation noted in late-stage embryonic Tcf-4 knockout mice (13). The Hedgehog pathway antagonizes Wnt signals as cells exit the crypt, allowing for proper terminal differentiation of the surface colonic epithelium (14). Transforming growth factor β/bone morphogenetic protein signaling also functions as a key negative regulator of proliferation and stem cell census: inhibition of bone morphogenetic protein signaling leads to ectopic crypt formation (15). How these and other signaling pathways are linked to and/or modified by specific cellular elements in the stem cell niche remains to be defined. Similarly, the cellular and molecular factors that determine ColEP responses to wound repair are unclear, although recent reports have emphasized the importance of the gut microbiota and Toll-like receptor-mediated pathways (16).

In this report, we use a model of epithelial injury produced by short-term oral administration of dextran sodium sulfate (DSS) to C57BL/6 (B6) mice. DSS treatment results in a rapid increase in epithelial permeability, followed by epithelial exfoliation and an immunoinflammatory response (17). There is a pronounced increase in ColEP proliferative activity in distal colonic crypts located next to areas of exfoliated epithelium. Our cellular and molecular analyses of this response in germ-free (GF) and conventionally raised (CONV-R) B6 wild-type and various knockout mice reveal a previously unappreciated function of macrophages: When recruited to a site of injury and activated by the microbiota, they support and promote proliferation of ColEPs. We propose that intestinal macrophages should be viewed as a crucial mobile cellular element that shapes the crypt progenitor cell niche in ways that allow ColEPs to mount an appropriate regenerative response to injury.

Materials and Methods

Mice. All experiments involving animals were performed according to protocols approved by the Washington University School of Medicine Animal Studies Committee. B6 wild-type and Rag-/- mice were rederived as GF by using protocols described in ref. 18. CONV-R B6 wild-type, Rag1-/-, Csf1op/op mice (all from The Jackson Laboratory), plus CONV-R B6 Myd88-/- animals (a gift from Emil Unanue, Washington University School of Medicine) were maintained free of specified pathogens in a barrier facility under a strict 12-h light cycle and fed the same autoclaved chow diet (B & K Universal, Yorkshire, U.K.) as their GF counterparts. Adult (6- to 10-week-old) male mice were used for all experiments.

A 2.5% aqueous solution of DSS (TDB Consultancy, Uppsala) was passed through a 0.22-μm cellulose acetate filter and then administered to GF and CONV-R mice for 7 d as drinking water. Control groups of mice received water alone.

Histochemical and Immunohistochemical Analyses. All analyses were performed in the 1- to 2-cm area of mucosa that bordered ulcers in the descending colon or in the corresponding area of the colon in untreated controls (n = 3–6 mice per group per experiment).

For morphometric and a subset of immunohistochemical studies, colons were dissected, placed in Bouins' fixative for 4 h at 24°C, and embedded in 2% agar for routine paraffin processing. Serial 5-μm-thick sections were cut perpendicular to the crypt-surface epithelial cuff axis and parallel to the cephalocaudal axis. Sections were stained with hematoxlyin and eosin (H+E) or periodic acid Schiff/Alcian blue (PAS/AB). For immunohistochemical studies, sections were deparaffinized in xylene, rehydrated in isopropanol, blocked in 1% BSA/0.3% Triton X-100, and incubated with the following Abs: Alexa Fluor 488-labeled rat anti-mouse F4/80 (macrophages; clone CI:A3–1, Caltag, Burlingame, CA); rabbit anti-pan-laminin (basement membrane; Sigma); Cy3-labeled rat anti-mouse smooth muscle actin (myofibroblasts; clone 1A4, Sigma); and rat anti-mouse CD31 (endothelial cells; clone MEC13.3, Becton Dickinson). Cycling cells were labeled in S phase by i.p. injection of a solution containing 120 mg/kg BrdUrd and 12 mg/ml 5′-fluor-2′-deoxyuridine (Sigma) 1 h before being killed. S-phase cells were identified in tissue sections by using goat anti-BrdUrd (19).

For another subset of immunohistochemical studies, colons were dissected and frozen in OCT compound. For direct immunofluorescence, 5-μm-thick sections were fixed in methanol (5 min at room temperature), blocked as above, and stained with Alexa Fluor 488-labeled Armenian hamster mAbs against CD3ε (T-cells; clone 145–2C11), FITC-labeled rat mAbs against B220 (B-cells; clone RA3–6B2), and Gr-1 (neutrophils; clone RB6–8C5). All these Abs were obtained from Becton Dickinson.

Laser Capture Microdissection (LCM) and GeneChip Analysis. Descending colons from DSS-treated mice were prepared for LCM by using protocols described in ref. 20. The pericryptal mesenchyme was harvested by using a PixCell IIe system and CapSure HS LCM caps (Arcturus, Mountain View, CA) (n = 3 mice per experimental group; 10,000 cells harvested per mouse). Total cellular RNA was extracted (PicoPure RNA isolation kit, Arcturus), and its quality was evaluated by using an Agilent (Palo Alto, CA) 2100 Bioanalyzer. Duplicate 5-ng aliquots of pooled intact RNA from each group were amplified and labeled (RiboAmp HS RNA Amplification Kit, Arcturus). The resulting biotinylated cRNA targets were used to probe MOE430A GeneChips (Affymetrix, Santa Clara, CA). LCM pericryptal mesenchyme-derived data sets and a previously generated data set that examined the effects of treating mouse macrophages for 3 h with LPS by using U74v2 GeneChips (21) were analyzed by using version 1.3 of dchip (22) and descisionsite 7.3 (Spotfire, Sommerville, MA) (see Results for further descriptions).

Results and Discussion

The ColEP Population Is Enhanced in Areas Adjacent to Ulcerated Wounds in Wild-Type Mice. Oral administration of 2.5% DSS for 7 d created small ulcers (<0.5 cm in greatest dimension) in the descending colons of all CONV-R adult B6 mice examined (n = 18). The adjacent 1- to 2-cm area of mucosa formed prominent “heaped borders” around these ulcers (Fig. 1 A and B). Crypts in this region were markedly elongated (mean height, 188 μm versus 90 μm for untreated mice; P < 0.01) (Fig. 1 A Inset and B Inset, H+E), and contained expanded zones of proliferation (Fig. 1 A Inset and B Inset, BrdU). EM analysis of the lower half of these elongated crypts showed a predominance of poorly differentiated columnar epithelial cells and a minor population of goblet lineage progenitors with very small amounts of mucin (Fig. 1B Inset, EM), consistent with previous morphologic descriptions of ColEPs detailed in the classic tritiated thymidine/EM autoradiographic studies of unperturbed normal adult mouse crypts by Chang and Leblond (2). Quantitative light and transmission EM studies showed that ColEPs represented 97% of the epithelial cells in the lower half of these crypts, compared with 49% in the crypts of untreated CONV-R controls. The control crypts contained equal numbers of fully differentiated goblet cells with large amounts of apical mucin comingled with ColEPs (Fig. 1 A Inset and B Inset, PAS/AB).

Fig. 1.
The intestinal microbiota and Myd88 signaling are necessary for the proper response to epithelial injury. (AF)H+E-stained sections of descending colon from adult B6 mice. DSS induces focal ulcer formation (dashed boxes). (A and B) Wild-type CONV-R ...

The Microbiota and Myd88 Are Necessary for the ColEP Proliferative Response to Injury. We analyzed the role of the microbiota in regulating the proliferative response of ColEPs to DSS-induced epithelial barrier injury by examining GF and CONV-R wild-type B6 mice and CONV-R Myd88-/- B6 mice. GF mice have no indigenous microbes, whereas My88-deficient mice are defective in signaling from Toll-like receptors that recognize a variety of microbial products (2325). Crypts from corresponding regions of the descending colons of untreated GF wild-type and CONV-R Myd88-/- mice contained crypts with proliferative zones that were similar in size and cellular census to those in untreated CONV-R wild-type controls (Fig. 1 C and E). After a 7-d treatment with DSS, the epithelium bordering descending colonic ulcers in GF wild-type and CONV-R Myd88-/- knockout mice appeared markedly attenuated, compared with untreated controls (Fig. 1 D and F). Crypts were significantly shorter [mean height, 46 μm (GF wild-type) and 75 μm (CONV-R Myd88-/-) (Fig. 1 D Inset and F Inset, H+E)]. Crypts also contained relatively fewer ColEPs and had an abundance of fully differentiated goblet cells (Fig. 1 D Inset and F Inset, PAS/AB and EM).

These changes correlated with marked decreases in ColEP proliferation, whether judged by qualitative assessment of the number of cells in S phase (Fig. 1 D Inset and F Inset, BrdU) or by quantifying M-phase cells; i.e., DSS produced significant ColEP hyperproliferation in CONV-R wild-type mice (4.6-fold increase compared with untreated CONV-R counterparts; P < 0.01), but statistically significant hypoproliferation in GF wild-type and CONV-R Myd88 -/- animals (4.5- and 2-fold decreases, respectively, compared with untreated controls; P < 0.01) (Fig. 1G). Note that the mean number of M-phase cells was not significantly different among the three groups of untreated mice (23–29 cells per 100 crypt sections). The observed changes in proliferation were not accompanied by changes in apoptosis (Fig. 1G). Therefore, we could attribute the hypoplastic, non-regenerative crypt phenotype in DSS-treated GF wild-type and CONV-R Myd88-/- mice to a lack of ColEP proliferation.

Toll-Like Receptor Signaling Through Myd88 in the Pericryptal Mesenchyme and Macrophages Is Necessary for Proper Epithelial Regeneration. Quantitative immunohistochemical studies showed that DSS treatment of CONV-R wild-type mice produced significant increases in mesenchymal macrophages (scored as F4/80-positive cells; 1.8-fold; P < 0.01), neutrophils (Gr-1-positive; 15.6-fold; P < 0.01), and lymphocytes (B220 and CD3ε-positive; 7.8-fold; P < 0.01) compared with untreated controls (Fig. 1H). DSS-treated GF and Myd88-/- mice showed smaller increases in neutrophils (5.3- and 1.2-fold, respectively) and lymphocytes (6.7- and 1.9-fold, respectively), but the increase in mesenchymal macrophages was equivalent to that observed in treated CONV-R wild-type animals (2.4- and 2.3 -fold, respectively; P < 0.01) (Fig. 1H). The average number of intraepithelial T lymphocytes per crypt was not significantly altered by DSS treatment or the absence of either Myd88 or the microbiota (mean of 0.5 cells per section in all cases).

The observed Myd88-dependence of ColEP proliferation indicated that the host injury response required detection of microbes and/or microbe-generated products. Most cell types in the colon contain Toll-like receptors, including the epithelial cells themselves (26). Immunohistochemical studies of CONV-R normal mouse colons showed that Myd88 was expressed in the epithelium and most underlying mesenchymal cells, including those that were components of the immune system (data not shown).

We tested the role of Myd88 signaling in the ColEP response to injury by examining chimeric animals. Irradiated CONV-R wild-type mice received bone-marrow transplants (BMT) (27) from either wild-type CONV-R mice (control group) or Myd88-/- mice (experimental group; n = 6 mice per group). Mice were examined 12 weeks after their BMT. Greater than 95% of macrophages in the colons of animals that had received bone marrow from Myd88 knockout donors were Myd88-negative, as judged by Ab staining of tissue sections (data not shown). In contrast, >95% of macrophages were Myd88-positive in the reconstituted control group that had received marrow from wild-type donors. ColEPs in reconstituted CONV-R wild-type mice demonstrated a hyperproliferative response to DSS-mediated injury similar to CONV-R wild-type mice that had not undergone irradiation and BMT (compare Figs. Figs.2A2A and and1G).1G). However, ColEPs in CONV-R chimeras with Myd88-/- bone marrow showed a hypoproliferative response to DSS injury that mimicked both GF wild-type and CONV-R Myd88 knockout mice. Myd88-/- bone marrow chimeras exhibited no statistically significant changes in epithelial apoptosis, whether or not they had received DSS (Figs. (Figs.1G1G and and2A2A).

Fig. 2.
Myd88-dependent ColEP response to wounding requires macrophages. (A) Quantification of epithelial proliferation (Left) and apoptosis (Right) in the descending colons of CONV-R B6 wild-type mice transplanted with bone marrow from either wild-type or Myd88 ...

The requirement for Myd88-competent, bone-marrow-derived cells for the colonic epithelial injury response led us to next evaluate mice that lacked specified components of the adaptive or innate immune system. Studies of CONV-R B6 Rag1-/- mice that lack mature B and T cells (n = 6 mice per group) (28) and CONV-R wild-type B6 mice treated with mAbs against Gr-1 to achieve a >95% ablation of their neutrophils (n = 6 mice per group) (29) revealed that neither of these cell populations was needed for the ColEP proliferative response to DSS-induced injury. However, analysis of CONV-R B6 Csf1op/op mice with >95% ablation of their colonic macrophages (n = 4 per group) (30) revealed a deficiency in the ColEP response that was similar to that encountered in GF wild-type and CONV-R Myd88 knockout animals (Fig. 2B).

Cellular and Molecular Evidence for Macrophage Activation in the Pericryptal Niche Associated with a Regenerative ColEP Response. Based on these findings, we examined the location and morphology of macrophages in the ColEP niche as a function of DSS treatment. In untreated CONV-R wild-type mice, F4/80-positive macrophages were concentrated in the lamina propria adjacent to the upper crypt and surface cuff epithelium (Fig. 3A). DSS treatment increased the presence of macrophages near the crypt base (Fig. 3B). Interestingly, these cells displayed multiple extended processes that were closely apposed to the basal surface of crypt base epithelial cells in areas of focal loss of laminin. Myofibroblasts and endothelial cells can also display cellular extensions around the crypt base in tissue sections (6). However, specific markers for these cell types (smooth muscle actin and CD31, respectively) did not colabel F4/80-positive macrophages in multilabel immunohistochemical studies, and neither cell type was significantly increased in number or exhibited changes in their distribution along the crypt under any of the conditions studied (n = 3 mice per group per treatment condition; e.g., Fig. 3 C and D). EM analysis confirmed that the mesenchymal cells with elongated processes were macrophages (Fig. 3 E and F). Immunohistochemistical and EM studies disclosed that the morphology of macrophages in DSS-treated GF wild-type and CONV-R Myd88-/- colons was quite different from that in CONV-R wild-type colons: In both cases, macrophages in areas of laminin loss had short processes that were unpolarized with respect to the basal surface of crypt base epithelial cells (Fig. 3 G and H; Myd88-/- not shown).

Fig. 3.
Activated macrophages are required for ColEP response to ulcerated injury. (A and B) Sections of descending colons stained with Alexa Fluor 488-labeled rat anti-F4/80 (green), rabbit anti-laminin, Alexa Fluor 594-labeled donkey anti-rabbit Ig (red), and ...

To assess the state of activation of macrophages, a GeneChip analysis was performed by using LCM pericryptal mesenchyme from GF and CONV-R Rag1-/- plus CONV-R Myd88-/- mice. We used Rag1-/- mice for this study because (i) their DSS-induced regenerative phenotypes were similar to GF and CONV-R wild-type animals and (ii) to eliminate the potentially confounding enrichment of mesenchymal lymphocytes observed in CONV-R wild-type mice (Fig. 1H). A data set of 205 transcripts was culled that had >1.5-fold enrichment in CONV-R Rag1-/- versus GF Rag1-/- and CONV Myd88-/- pericryptal mesenchyme (this threshold cutoff value is based on the commonly used lower confidence bound of 1.3, see ref. 22 for discussion) (the list of 205 transcripts can be found in Table 1, which is published as supporting information on the PNAS web site).

Comparing LCM CONV-R Rag1-/- mesenchyme to the other two populations eliminated transcripts enriched only by the presence of microbes (CONV-R Rag1-/- versus GF Rag1-/- comparison) as well as lymphocyte-specific genes (CONV Rag1-/- versus Myd88-/- comparison).

To mine this 205-member data set for transcripts enriched in activated macrophages, we compared it with a second data set developed by using the same computational constraints but derived from a previously published compendium of global gene expression in primary mouse macrophages activated by LPS in vitro (21). Of the 205 genes, 104 were represented by probe sets in the earlier generation GeneChip used for the in vitro macrophage activation study. Transcripts from 41 of the 104 “overlapping” genes (39%; see Table 1) were enriched at least 1.5-fold in LPS-activated macrophages (given the sizes of the data sets, an overlap of even a single gene would have a P value of <0.001). The 41 transcripts included those encoding cytokines (chemokine, C-X-C motif, and ligands 2 and 10), acute phase reactants (metallothioneins 1 and 2 and ceruloplasmin) and IFN-γ induced genes (numerous GTPases known to dominate this response, Gbp2, Gbp3, Iigp, Tgtp, and Gtpi) (31). Connexin 43, which is thought to coordinate activated macrophage responses by formation of gap junctions (32), was also represented. Interestingly, Cox-2 (cyclooxygenase 2), a therapeutic target for preventing familial colorectal neoplasms associated with mutations in APC (adenomatous polyposis coli), a component of the Wnt/β-catenin pathway (33), was also present.

Molecular Mediators of the ColEP Response Emanating from Pericryptal Mesenchyme. The 205-member data set of transcripts enriched in mesenchyme surrounding regenerative crypts was searched for candidate molecular mediators of the ColEP response. We used the current version of automated gene ontology (GO) terms (www.geneontology.org) to find genes with the identifiers “extracellular space,” “basement membrane,” “extracellular matrix” and “integral to plasma membrane.” In the data set, 59 genes (28%) were identified by at least one of these terms (Table 1). This analysis yielded a category of genes encoding secreted factors. Many are known to enhance proliferation: e.g., Cox-2, which modulates proliferation through effects on prostaglandin synthesis; insulin-like growth factor-1 (Igf-1) and interleukin 11 (Il-11), which are known to play roles in the adaptation to intestinal failure (34); Wnt4 (1113); and Reg3β and Reg3γ, which enhance proliferation of transformed colonic epithelial cell lines (35), although recent in vivo studies suggest their primary roles may be to limit inflammation (36).

A second, well represented group of genes affects extracellular matrix and basement membrane function and stability. Timp3 (tissue inhibitor of metalloproteinase 3) inhibits matrix metalloproteinases, four of which were present in our data set (Mmp13, Adam9, Adamts1, and Adamts4). These enzymes degrade extracellular matrix components, among their other proinflammatory functions (37). Sparc (osteonectin) and genes encoding heparin sulfate proteoglycans, including Col18A1 (collagen XVIII), Sdc1 (syndecan 1), Kera (keratocan), and Cspg2 (chondroitin sulfate proteoglycan 2), are part of the basement membrane and well known repositories for growth factors, such as Wnts (38). Collagen XVIII is also a binding site for l-selectin (also in our data set), which could mediate binding of activated macrophages to the basement membrane underlying ColEPs (39). These findings emphasize the integrated nature of the cellular and molecular communications that appear to underlie the regenerative response of ColEP response to DSS-induced injury: macrophage influx, activation, and apposition to epithelial progenitors leads to production of growth factors whose bioavailabilty, in turn, may be modulated through effects on basement membrane components.

Prospectus. Macrophages do not appear to be necessary for normal maintenance of crypts, because Csf1 op/op colonic crypts have normal morphology (30). Nonetheless, our data indicate that activated macrophages in the ColEP niche orchestrate the enrichment of transcripts encoding a number of factors that promote proliferation and survival of epithelial progenitors during injury.

Macrophages and dendritic cells are able to sample microbial components of the intestinal lumen, providing a key link between the microbiota and epithelial barrier functions (4042). A recent report by Rakoff-Nahourn et al. (16) demonstrated the importance of Toll-like receptors and the intestinal microbiota in a similar DSS injury model. Our results with gnotobiotic-treated mice rather than antibiotic-treated mice support their conclusions. In addition, our results reveal a previously unrecognized function for macrophages, namely a mobile cellular constituent of a dynamic colonic epithelial stem cell niche.

Finally, the functional genomics approach can be used to characterize the molecular features of epithelial progenitors in their gastric, small intestinal, and colonic niches (www.scgap.org) (19, 43). ColEPs amplified during the regenerative response to DSS-mediated injury now provide a rich source of normally rare crypt progenitors for further molecular characterization. Such characterization should help provide better understanding of the shared and distinctive biological properties of adult gut epithelial progenitors and of their adaptive responses to physiological and pathological changes in their environment.

Supplementary Material

Supporting Table:

Acknowledgments

We thank Peter Murray (St. Jude Children's Research Hospital, Memphis, TN) and Emil Unanue for reagents and Scott Lovitch, Kathy Frederick, Laura Mandik-Navak, Fei Shi, Paul Allen, Ana Mendoza, Olle Vidal, Jamie Dant, David O'Donnell, and Maria Karlsson for helpful discussions and technical assistance. This work was funded in part by National Institutes of Health Grants DK02954, DK63483, and DK06220.

Notes

Author contributions: T.S.S. designed research; S.L.P. and T.S.S. performed research; S.L.P., J.M.D., J.C.M., J.I.G., and T.S.S. analyzed data; and J.C.M., J.I.G., and T.S.S. wrote the paper.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: ColEP, colonic epithelial progenitor; DSS, dextran sodium sulfate; B6, C57BL/6; GF, germ-free; CONV-R, conventionally raised; H+E, hematoxlyin and eosin; PAS/AB, periodic acid Schiff/Alcian blue; LCM, laser capture microdissection.

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