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First published online 16 February 2005
doi: 10.1242/dev.01700

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Crypt-restricted proliferation and commitment to the Paneth cell lineage following Apc loss in the mouse intestine

¹ Institut Cochin, INSERM U567, CNRS UMR8104, Université Paris V, 24 rue du Fb St-Jacques, 75014 Paris, France
² UMR-S INSERM U490, 45 rue des St-Pères, 75006 Paris, France
³ INSERM U434, 27 rue Juliette Dodu, 75010 Paris, France
⁴ Institut Curie, UMR 144, 75005 Paris, France

View larger version (49K): [in a new window]   Fig. 1. TAM injection induces epithelium-specific inactivation of Apc all along the crypt-villus axis. (A) Structure of the conditionally targeted allele of Apc. Exons 11 to 15 are indicated as black bars. The positions of the primers used for the detection of each allele are indicated: 13F, 15R. In floxed mice, induction of Cre recombination by TAM injection induced exon 14 deletion, generating a frameshift mutation at codon 580 (appearance of the stop codon TAA). (B, left) Cre expression in the small intestine before (D0) and 2 days after (D2) TAM injection in mutant mice. (Middle) RT-PCR analyses on days 0 (D0), 2 (D2) and 4 (D4) in mutant mice reveal the floxed (Apclox) and deleted (Apcex14) Apc alleles. (Right) Immunostaining of APC on day 4 in control and mutant mice, showing loss of APC protein in the mutant mice.

View larger version (112K): [in a new window]   Fig. 2. Loss of Apc along the crypt-villus axis induces progressive crypt extension associated with intense proliferation. (A) Representative Hematoxylin and Eosin (H&E)-stained sections of jejunum from control (left) and mutant mice (right) at day 4, shown at low and high magnification. (B) Representative H&E-stained sections of proximal colon from mutant mice (day 4), shown at low, medium and high magnification. Line indicates the junction between dysplastic (top section) and normal areas (bottom section). Arrows and arrowheads indicate mitotic and apoptotic cells, respectively. (C) Representative H&E- (top) and Ki-67- (bottom) stained sections of jejunum from control (day 4, D4) and mutant (D2, D3, D4) mice. Abnormal crypt extension and similar Ki67 staining were observed all along the small intestine from the duodenum to the ileum in the mutant mice. Brackets indicate the dysplastic zone.

View larger version (108K): [in a new window]   Fig. 3. Apc deficiency activates apoptosis in the crypts and slows down cell migration. (A) High magnification images of H&E-stained sections and sections immunostained for active caspase 3 from control and mutant mice (D4). Arrows indicate apoptotic cells. (B) BrdU staining at 2 and 24 hours after injection in control and mutant mice. Brackets indicate the position of the BrdU-stained cells that have migrated in the villi in 24 hours.

View larger version (100K): [in a new window]   Fig. 4. Distinct profiles of ß-catenin localization associated with distinct profiles of Myc and cyclin D1. (A)ß-Catenin immunostaining of jejunum sections from control and mutant mice (D4), shown at low and high magnification. (B, left) Levels of Myc and cyclin D1 transcripts in the jejunums of control and mutant mice on day 4, as measured by RT-PCR. The level of each transcript is expressed relative to that in wild-type mice. (B, right) Western blot analysis of ß-catenin, Myc and Cyclin D1 in the jejunums of control and mutant mice on day 4. (C) Myc (a,c) and Cyclin D1 (b,d) staining in sections of jejunum from control (a,b) and mutant mice (c,d) on day 4. Similar staining patterns were observed all along the small intestine.

View larger version (101K): [in a new window]   Fig. 5. Differentiated lineage analysis in intestinal mucosa: commitment to the Paneth cell lineage. (A) Villin, UEA-1 and chromogranin staining in sections from control and mutant mice on day 4. Arrows indicate chromogranin-positive cells. (B) H&E and ß-catenin staining of the crypt compartment of control mice (a,f). Arrows indicate Paneth cells (a) and nuclear ß-catenin staining in the Paneth cells (f). In situ hybridization of lysozyme and cryptdins in the small intestine of control mice (b,g), in colon polyps from APC+/- mice at low (c,h) and high magnification (d,i), and in the small intestine of mutant mice (e,j) at day 4.

View larger version (23K): [in a new window]   Fig. 6. Cryptdin/defensin family genes are direct targets of ß-catenin signaling. (A) Consensus Tcf-binding sites (black boxes) are present in 300 bp regions of promoter sequences of distinct cryptdin and defensin genes. (B, left) Schematic representation of cryptdin/defensin promoter constructs showing the putative Tcf-binding sites (black boxes). (B, right) Responses of various promoter fragments (cryptdin5-2500, HD5-1000 and HD6-2500) to activated ß-catenin and Tcf4 in 293T cells (*P<0.05). (C) HD6 promoter constructs of various lengths showing the putative Tcf-binding sites (left), mutated or not, and their response to ß-catenin/Tcf4 (right, *P<0.05).

View larger version (14K): [in a new window]   Fig. 7. Induction of HD6 in human cancers. Levels of Myc, cyclin D1 and HD6 transcripts in 10 human colon cancers (A-J) and matched normal mucosa samples, as measured by quantitative RT-PCR. Results are expressed as a ratio of the levels in tumors to in their normal counterparts.