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. 2012 Mar 30;149(1):146-58.
doi: 10.1016/j.cell.2012.02.042.

The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor

Anne E Powell  1 , Yang Wang, Yina Li, Emily J Poulin, Anna L Means, Mary K Washington, James N Higginbotham, Alwin Juchheim, Nripesh Prasad, Shawn E Levy, Yan Guo, Yu Shyr, Bruce J Aronow, Kevin M Haigis, Jeffrey L Franklin, Robert J Coffey
Affiliations

The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor

Anne E Powell et al. Cell. .

Abstract

Lineage mapping has identified both proliferative and quiescent intestinal stem cells, but the molecular circuitry controlling stem cell quiescence is incompletely understood. By lineage mapping, we show Lrig1, a pan-ErbB inhibitor, marks predominately noncycling, long-lived stem cells that are located at the crypt base and that, upon injury, proliferate and divide to replenish damaged crypts. Transcriptome profiling of Lrig1(+) colonic stem cells differs markedly from the profiling of highly proliferative, Lgr5(+) colonic stem cells; genes upregulated in the Lrig1(+) population include those involved in cell cycle repression and response to oxidative damage. Loss of Apc in Lrig1(+) cells leads to intestinal adenomas, and genetic ablation of Lrig1 results in heightened ErbB1-3 expression and duodenal adenomas. These results shed light on the relationship between proliferative and quiescent intestinal stem cells and support a model in which intestinal stem cell quiescence is maintained by calibrated ErbB signaling with loss of a negative regulator predisposing to neoplasia.

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Figures

Figure 1
Figure 1. Lineage tracing in the small intestine and colon confirms Lrig1 marks SCs
(A-C) Generation of Lrig1-CreERT2/+ mice. (A) Schematic representation of the Lrig1-CreERT2 targeting vector. A tamoxifen-inducible Cre (CreERT2) was targeted into the translational initiation site of the endogenous Lrig1 locus. Southern blot analysis of embryonic SCs with 3′, 5′ and internal neo probes confirmed the correct integration at a frequency of 8.7% (B and data not shown). Chimeras were mated with FlpE mice to achieve germ-line transmission and neo cassette removal. The resulting heterozygous and homozygous mice were viable and fertile. (C) Lrig1-CreERT2 animals were genotyped by specific Lrig1-CreERT2 PCR. (D0-G0) Low-power view of Lrig1-CreERT2/+;R26RLacZ/+ lineage-labeled small intestine at different time points following a single i.p. injection of 2mg tamoxifen. (D1-G1) Representative sections of high-power view of β-gal+ small intestine. (H0-K0) Low-power view of Lrig1-CreERT2/+;R26RLacZ/+ lineage-labeled colon at different time points following a single i.p. injection of 2mg tamoxifen. (H1-K1) Representative sections of high-power view of β-gal+ colonic crypts. Scale bars represent 100μm in D0 and H0; 200μm in E0-G0 and I0-K0; 50μm in D1-G1 and 25μm in H1-K1. See also Figure S1 and Table S1-S2.
Figure 2
Figure 2. β-gal labels Lrig1+ cells in the SC niche, persists in the long-term and labeling differs in Lgr5-reporter mice
(A) One day after tamoxifen induction, 50% of small intestinal crypts and 40% of colonic crypts are labeled. At 90 days, the number of labeled crypts decreases to 18% in the small intestine and 10% in the colon. (B-C) Frequency of β-gal+ cells at different positions relative to the small intestinal (B) and colonic (C) crypt base one day after tamoxifen administration. (D-I) Co-staining with various differentiation markers to confirm multipotency of progeny of β-gal+ cells: Mucin2 (Muc 2) marks goblet cells (D,G); Chromogranin-A (Chr A) marks enteroendocrine cells (E,I); Lysozyme (Lys) marks Paneth cells (F); and Carbonic Anhydrase IV (CAIV) marks enterocytes (H). (J-M) Whole-mount view of Lrig1-CreERT2/+;R26RLacZ/+ and (N-Q) Lgr5-EGFP-IRES-CreERT2;R26RLacZ/+ lineage-labeled intestines. Error bars represent s.e.m. Scale bars in D-I represent 25μm and J-Q represent 500μm. See also Figure S2.
Figure 3
Figure 3. Expression patterns of Lrig1 and Lgr5 in adult mouse colon by immunofluorescence and FACS
(A-B) Immunofluorescent detection and confocal microscopy of cross-sections in Lgr5-EGFP-IRES-CreERT2 mouse colon detected a subset of Lrig1+ cells (red) present at the crypt base, distinct from Lgr5+ cells (green). (C) Relative position of Lrig1+ and Lgr5+ populations by immunofluorescence for Lrig1 and direct EGFP fluorescence of the Lgr5 reporter. (D) FACS analysis of Lgr5-EGFP-IRES-CreERT2 mouse intestinal epithelial cells (n=9); Lrig1 was detected in 2.4% of total enterocytes and 4.8% of total colonocytes. Using EGFPhi as a marker of Lgr5 expression, 0.5% of total enterocytes and 0.2% of total colonocytes were Lgr5+;Lrig1+. (E) A representative colonic crypt stained for Lrig1 (red) and Ki67 (white). (F) Twenty-five percent of Lrig1+ cells also stained with Ki67, whereas 75% of the Lgr5+ population also stained with Ki67. (G) BrdU incorporation, measured in Lrig1 mouse colonocytes by FACS. Lrig1+;BrdU+ cells were not detected in uninjected mice; 7% of Lrig1+ colonocytes co-expressed BrdU 2 hours after injection (n=3). Mice injected daily for five days and examined six weeks later, had 12% Lrig1+;BrdU+ colonocytes (n=3). “LT” indicates long-term, label-retaining experiment. Scale bars represent 25μm (A-B, E). Error bars represent s.e.m. See also Figure S3-S4.
Figure 4
Figure 4. Lrig1+ cells can be traced from birth and proliferate in response to irradiation damage
(A) Lrig1-CreERT2/+;R262RLacZ/+ mice injected with tamoxifen at postnatal day one and sacrificed six weeks later, harbored single, β-gal+ label-retaining colonocytes at the crypt base. (B) Examination of β-gal in the p1 injected animals revealed 7% colonic crypts (n=2; 400 crypts/animal) were entirely β-gal+, while nearly 13% contained single, β-gal+ label-retaining colonocytes at the crypt base. (C) In long-term, tamoxifen-induced Lrig1-CreERT2/+;R262RLacZ/+ mice, 8% of β-gal+ cells were single cells at the base of the colonic crypt. (D-E) One week after irradiation damage, many colonic crypts contained β-gal+ cells in clusters. (F) Clusters of β-gal+ cells increased in animals subjected to irradiation. In control (unirradiated) animals, 5% of colonic crypts contained β-gal+ clusters; this number increased to 12% in irradiated animals. (G) Examination of β-gal+ cells in control and irradiated Lrig1-CreERT2/+;R262R-LacZ animals revealed that 14% of β-gal+ cells stained with the proliferative marker Ki67 in control animals, whereas this number increased to 55% in irradiated animals. All scale bars represent 25μm. Error bars represent s.e.m. See also Figure S3-S4.
Figure 5
Figure 5. RNA-Seq analysis of Lrig1+ and Lgr5+ cells
(A). Gating strategies for FACS analysis of Lgr5-EGFP-IRES-CreERT2 mouse colonic epithelial cells (n=5) used to isolate Lrig1+ and Lgr5+ populations (Lrig1-Alexa568 and Lgr5-EGFPhi, respectively) for RNA-Seq analysis. (B) Scatter plot depicting representative gene expression profiles of Lrig1+ (Y-axis) and Lgr5+ (X-axis) cells. Fragments Per Kilobase Per Million reads (FPKM) analysis was conducted and ~21,000 genes are shown in the plot. Transcripts detected below 0.5 FPKM (little biological significance) are shown in gray. Red indicates genes significantly expressed in Lrig1+ cells, compared to Lgr5+ cells, while blue indicates genes that are significantly expressed in Lgr5+, compared to Lrig1+ cells. Transcripts shared by both Lgr5+ and Lrig1+ cells are shown in green. Approximately 2,500 significantly differentially regulated genes are shown (red and blue). Some of genes discussed in Results are annotated in the figure. Genes plotted between 100 and 1000 FPKM are highly expressed. (C) Abstract network depicting Lgr5- and Lrig1-associated genes in each list that share structural or functional associations, including gene ontology properties, human disease and mouse knockout phenotype associations, shared regulatory elements and protein interactions. Significant gene feature associations that are unique or shared between the two cell compartment gene lists are shown. “TFBS” stands for a multispecies conserved transcription factor binding site. See also Figure S5 and Table S3.
Figure 6
Figure 6. Lrig1-CreERT2/+;Apcflox/+ mice develop colonic tumors
(A) Schematic depiction of experimental design. Mice were injected with tamoxifen daily for three days, monitored by colonoscopy starting at day 50 and sacrificed 100 days post-tamoxifen induction. (B-C) Examination of mice three months post-induction revealed the mice developed distal colonic tumors (shown by colonoscopy in B and by whole mount in C). (D) H&E staining of a tissue cross-section from a representative colon. Individual tumors are indicated by the asterisks. (E) Distribution and multiplicity of tumors from seven mice with the greatest number of adenomas formed in the jejunum. (F) High-grade dysplasia was present within a large tumor (black box in D). (G-I) Immunohistochemical examination of colonic tumors for Ki67 expression (brown in G), nuclear and cytosolic β-catenin (brown in H), and high-power image of a gland that contains both cytosolic- and membrane-associated β-catenin (I). (J) Immunofluorescent examination of colonic tumors for Apc expression (white); Apc expression is heterogeneous. Scale bars represent 50μm in F, 25μm in G, I and J and 75μm in H. Error bars represent s.e.m. See also Figure S6.
Figure 7
Figure 7. Homozygous Lrig1-CreERT2 mice exhibit up-regulation of ErbB1-3 and develop duodenal adenomas and superficially invasive carcinomas
(A) Representative western blot showing increased ErbB1-3 and pErk1/2 levels in crypt epithelium isolated from small intestine of Lrig1-CreERT2/CreERT2 (Cre/Cre) mice compared to wild-type (WT) mice. (B) Quantification of protein expression levels shown in (A). (C-D) Immunohistochemical examination of ErbB2 (brown) in intestinal crypts from WT (C) and Cre/Cre (D) mice. Black brackets indicate differential staining at the crypt base. (E) Representative H&E staining of an adenoma from a five-month-old Lrig1-CreERT2/CreERT2 mouse. Tumors exhibited low-grade dysplasia and a marked plaque-like expansion of Brunner’s glands (n=14). (F) One tumor exhibiting histological progression: areas of cribiform architecture and loss of cellular polarity and focal extension of neoplastic glands into deeper layers of the bowel wall, suggestive of early invasion (inset). (G) Representative western blot showing increased ErbB1-3 and pErk1/2 levels in tumor (T) compared to gross normal duodenum (N) of Lrig1-CreERT2/CreERT2 mice. (H-I) Immunohistochemical examination of pErk1/2 (brown in H) in tumors from Lrig1-CreERT2/CreERT2 mice; boxed region magnified in I. (J-K) Immunohistochemical examination of ErbB2 (brown in K) in tumors from Lrig1-CreERT2/CreERT2 mice; boxed region magnified in J. Scale bars represent 25μm in C, D, I and J, represent 100μm in E and F and 75μm in (H and K). Error bars represent s.e.m. See also Figure S7 and Table S4.
See this image and copyright information in PMC

Comment in

  • Stem cells: Marking stem cells.
    Alderton GK. Alderton GK. Nat Rev Cancer. 2012 May 24;12(5):318. doi: 10.1038/nrc3268. Nat Rev Cancer. 2012. PMID: 22525573 No abstract available.

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