Loss of homeostasis in small intestines of <i>vABKO a</i>nd <i>vTKO</i> mice.

<p>(<b>A</b>) Mice were injected with tamoxifen for five consecutive days and then sacrificed 3 days after the final injection. Small intestines were isolated and length determinations were made. Small intestine lengths were normalized to body weights, which were determined prior to the first tamoxifen-injection. Data is presented as mean +/− SEM. Asterisk (*) indicates significantly different after tamoxifen injection as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. The actual P-values are 0.38 (WT), 0.76 (<i>vil-Cre-ER^T2</i>), 0.003 (<i>vAKO</i>), 0.31 (<i>vBKO</i>), 0.002 (<i>vABKO</i>), 0.46 (<i>vACKO</i>) and 0.004 (<i>vTKO</i>). The small intestinal lengths of <i>vAKO</i>, <i>vABKO</i> and <i>vTKO</i> mice were significantly different from WT mice injected with tamoxifen as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. Actual P-values are 0.40 (<i>vil-Cre-ER^T2</i>), 0.03 (<i>vAKO</i>), 0.10 (<i>vBKO</i>), 0.00006 (<i>vABKO</i>), 0.36 (<i>vACKO</i>) and 0.0006 (<i>vTKO</i>). (<b>B</b>) Duodenums isolated from mice treated as described in A were photographed under a dissection microscope. Scale bar: 0.5 mm. (<b>C</b>) Significant shortening of villi in small intestines of <i>vABKO</i> and <i>vTKO</i> mice. Length of individual villi shown in panel B were measured (30 villi per mouse). Data is presented as mean +/− SEM. Asterisk (*) indicates significantly different after tamoxifen injection as determined by a Student's t-test. P-values are 0.00008 (<i>vABKO</i>) and 0.007 (<i>vTKO</i>). Villi lengths of <i>vil-Cre-ER^T2</i>, <i>vABKO</i> and <i>vTKO</i> mice were significantly different from WT mice injected with tamoxifen as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. Actual P-values are 0.001 (<i>vil-Cre-ER^T2</i>), 0.27 (<i>vAKO</i>), 0.45 (<i>vBKO</i>), 0.0005 (<i>vABKO</i>), 0.25 (<i>ACKO</i>) and 0.03 (<i>vTKO</i>).</p

Enhanced Wnt-signaling and differentiation of CBC cells in <i>vABKO</i> and <i>vTKO</i> mice.

<p>(<b>A</b>) Tissue sections of small intestines prepared from <i>Vil-Cre-ER^T2</i> (left panel), <i>vABKO</i> (middle panel) and <i>vTKO</i> (right panel) mice were stained with antibodies specific for beta-catenin (green). Nuclei were stained with hematoxylin (blue). Green hatched lines outline nuclei. Scale bar: 20 µm. (<b>B</b>) Quantitation of nuclear beta-catenin staining is shown in A. Data is presented as mean +/− SEM. Asterisks in panel B indicate significantly different from vil-Cre-ER^T2 mice as determined by a Student's t-test. (<b>C</b>) Intestinal sections were stained with Periodic Acid Schiff/alcian blue to label Paneth cells and goblet cells from <i>Vil-Cre-ER^T2</i> (left panel), <i>vABKO</i> (middle panel) and <i>vTKO</i> (right panel) mice. Insets are magnifications of boxed regions shown in middle and right panel. Asterisks in left panel indicate CBC cells. Asterisks in middle and right panels indicate immature Paneth cells. Arrows in middle and right panel indicate small granules of immature Paneth cells. Scale bar: 10 µm.</p

Targeted disruption of <i>Cdc25B</i> in mice.

<p>(<b>A</b>) Structure of targeting vector and chromosomal organization of <i>Cdc25B</i> locus before and after Cre-mediated excision. The genomic organization of the mouse <i>Cdc25B</i> gene was disrupted by inserting into intron 1 the neomycin phosphotransferase cDNA driven by the phosphoglycerine kinase promoter (pGK-neo) as a selectable marker. Exons are represented by black boxes. The location of Hind III (H), Bam HI (B) and Kpn I (K) site is indicated and <i>loxP</i> sites are represented by yellow triangles. Sizes of upstream (3.3 kb) and downstream (4.5 kb) homologous arms are indicated. Position of probes used for Southern blotting are shown. Red triangles depict the locations of PCR primers used for genotyping. Abbreviations: +, wild type allele; R, recombinant allele; F, floxed allele; WT, wild type. (<b>B</b>–<b>C</b>) Southern blot analysis demonstrating homologous recombination in the <i>Cdc25B</i> locus. ES cell genomic DNA was digested with Hind III (B) and Bam HI (C), and Southern blotting was performed using the 5′ and 3′ probes shown in panel A. The genotype of each ES cell line is indicated. The location of size markers is shown on the left. (<b>D</b>) Southern blot analysis demonstrating Cre-mediated recombination in the <i>Cdc25B</i> locus. ES cell clones containing the recombinant allele were expanded and transiently transfected with a plasmid encoding Cre recombinase. Genomic DNA was digested with Kpn I (K), and Southern blotting was performed using the internal probe shown in panel A. The genotype of each ES cell line is indicated. Location of size markers is shown on left. (<b>E</b>) PCR analysis of mouse tail DNA. Mouse tail DNA was amplified with PCR primers depicted as red triangles in panel A. The wild type (+) allele produced a 383 bp PCR product and floxed allele (F) produced a 433 bp PCR product. The genotype of each mouse is indicated. The location of size markers is shown on the right.</p

Significant shortening of large intestine of <i>vABKO</i> and <i>vTKO</i> mice.

<p>Mice were injected with tamoxifen for five consecutive days and then sacrificed 3 days after the final injection. Large intestines were isolated and length determinations were made. Large intestine lengths were normalized to body weights, which were determined prior to the first tamoxifen-injection. Data is presented as mean +/− SEM. Asterisks indicate significantly different after tamoxifen injection as determined by a Student's t-test. P-values are 0.059 (WT), 0.32 (<i>vil-Cre-ER^T2</i>), 0.020 (<i>vAKO</i>), 0.27 (<i>vBKO</i>), 0.0009 (<i>vABKO</i>), 0.12 (<i>vACKO</i>) and 0.003 (<i>vTKO</i>). Large intestinal lengths of <i>vABKO</i>, <i>vACKO</i> and <i>vTKO</i> mice were significantly different from WT mice injected with tamoxifen as determined by a Student's t-test. P-values are 0.94 (<i>vil-Cre-ER^T2</i>), 0.82 (<i>vAKO</i>), 0.30 (<i>vBKO</i>), 0.00034 (<i>vABKO</i>), 0.041 (<i>vACKO</i>) and 0.0077 (<i>vTKO</i>).</p

Tamoxifen injection induced efficient recombination within <i>Cdc25A</i> and <i>Cdc25B</i> loci.

<p>(<b>A</b>) Genomic DNA isolated from the small and large intestines of tamoxifen-treated mice were assessed for Cre-mediated excision by Southern blotting. Deletion frequencies are shown below each lane and were determined by measuring band intensities using a Molecular Dynamics Storm imager. (B) Total RNA isolated from the small intestine (jejunum) of tamoxifen-treated WT and <i>vB^f/−</i> mice was reverse-transcribed into cDNA. qRT-PCR was used to determine relative amounts of <i>Cdc25B</i> mRNA. The data is presented as mean +/− SEM. Asterisk (*) indicates significantly different from WT, P  =  0.012 by Student's t-test. Note that <i>vB^f/−</i> mice are generated by crossing <i>Cdc25B</i> null mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015561#pone.0015561-Lincoln1" target="_blank">[26]</a> with <i>Cdc25B</i> conditional mice. The PCR primers detect transcript arising from the null allele but not the deleted floxed allele. Thus, a 50% decrease in relative expression indicates complete loss of <i>Cdc25B</i> expression.</p

Response of small intestinal epithelial cells to irinotecan.

<p>(<b>A</b>) Significant decrease in the number of BrdU postive cells in the small intestinal crypts of irinotecan-treated mice. WT mice were injected with PBS (cont) or irinotecan (CPT) for 6 consecutive days and sacrificed on day 7. One hour prior to sacrifice, mice were injected with BrdU. Intestines were isolated and sections were stained with an antibody specific for BrdU. Dotted lines demark crypt margins. Red: BrdU, Blue: nuclei (DAPI). Scale bar: 10 µm. (<b>B</b>, <b>C</b>) Significant loss of crypts in the small intestines of irinotecan-treated mice. Intestines were isolated from irinotecan-treated mice (B) and <i>TKO</i> mice (C), and sections were stained with H & E. Arrows in (B) indicate remaining crypts. Scale bar: 50 µm. (<b>D–F</b>) Irinotecan induces significant apoptosis in the crypts of treated mice at early time points after irinotecan-treatment. (D) Intestinal sections were prepared after two irinotecan administrations and stained with H & E. Arrows depict apoptotic cells. Scale bar: 10 µm. (E) Intestinal sections from WT mice treated with PBS (cont)) and irinotecan (CPT) were stained by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Green: TUNEL, Blue: nuclei (DAPI). Scale bar: 100 µm. (F) Intestinal sections from WT mice treated with PBS (left) and irinotecan (right) were stained for cleaved caspase-3. DAB (3, 3′-diaminobenzidine) was used as a substrate (brown) and sections were counter-stained with hematoxylin (blue). Scale bar: 50 µm. (<b>G</b>) Differentiation along the enterocyte lineage is not affected in irinotecan-treated mice. Intestines isolated from PBS- or irinotecan-treated WT mice were stained with the enterocyte marker, L-Fabp. Dotted lines demark crypt margins. Green: L-Fabp, Blue: nuclei (DAPI). Scale bar: 10 µm. (<b>H</b>) Loss of goblet cells in irinotecan-treated mice. Intestinal sections were stained with Periodic Acid Schiff/alcian blue to label goblet cells (indicated with arrow). Sections from WT (left), <i>TKO</i> (center) and irinotecan-treated WT mouse (right) are shown. Scale bar: 20 µm. (<b>I</b>) Significant infiltration of neutrophils in crypts of irinotecan-treated mice. The numbers of neutrophils were counted in 10 randomly chosen microscopic fields at 40X magnification in untreated WT, <i>TKO</i> and irinotecan-treated WT mice (CPT). Asterisks indicate significantly different from WT as determined by Student's t-test. WT and <i>TKO</i>: not significant, P = 0.33; WT and CPT: significant, P = 0.0006; <i>TKO</i> and CPT: significant, P = 0.02.</p

Proliferation and apoptosis in crypts of <i>Cdc25</i>-disrupted mice.

<p>(<b>A</b>) Mice were injected with tamoxifen for five consecutive days and then sacrificed 3 days after the final injection. One hour prior to sacrifice, mice were injected with BrdU. Intestines were isolated, and sections were stained with BrdU antibody (brown) and counterstained with hematoxylin (blue). Scale bar: 20 mm. (<b>B</b>) Mice were injected with tamoxifen for five consecutive days and were sacrificed 3 days after last tamoxifen injection. Intestines were isolated and sections were stained for cleaved caspase-3. 3, 3′-diaminobenzidine (DAB, brown) was used as a substrate, and sections were counter-stained with hematoxylin. Arrows indicate cells at the tip of villi, which stain positive for cleaved caspase-3. Scale bar: 0.1 mm. (<b>C–D</b>) Crypts within the proximal portion of the small intestine of tamoxifen treated mice were examined for mitotic cells (presence of mitotic figures) (C) and apoptotic cells (presence of fragmented nuclei) (D). Areas containing Brunner's gland were excluded from analysis. Twenty crypts were counted per mouse and three mice of each genotype were evaluated. Similar patterns were observed in mid and distal portions of the small intestine (data not shown). Data is presented as mean +/− SEM. Asterisks in panel C indicate significantly different from WT mice as determined by a Student's t-test (M-phase cells). *, P<0.05; **, P<0.01; ***, P<0.001. Actual P-values are 0.22 (<i>vil-Cre-ER^T2</i>), 0.045 (<i>vAKO</i>), 0.16 (<i>vBKO</i>), 0.00056 (<i>vABKO</i>), 0.17 (<i>ACKO</i>) and 0.00055 (<i>vTKO</i>). Asterisks in panel D indicate significantly different number of apoptotic cells from <i>WT</i> mice as determined by a Student's t-test. P-values are 0.48 (<i>vAKO</i>), 0.33 (<i>vBKO</i>), 0.0011 (<i>vABKO</i>), 0.064 (<i>ACKO</i>) and 0.0010 (<i>vTKO</i>). (<b>E</b>) Mice were injected with tamoxifen as described in A and small intestines were isolated, sectioned and stained for inactive Cdk1 (phosphorylated on Tyr-15, brown) and counterstained with hematoxylin (blue). Arrows indicate epithelial cells positively stained with the phospho-Tyr 15 CDK1 antibody. Scale bar: 20 µm.</p

Differentiation of CBC cells into immature Paneth Cells in <i>Cdc25</i>-disrupted mice.

<p>Small intestines were dissected and processed for Transmission Electron Microscopy. EM sections of crypts from <i>vil-Cre-ER^T2</i> (A), <i>vABKO</i> (B, C) and <i>vTKO</i> (D, E) mice. Insets in panels B and D are shown at higher magnification in panels C and E, respectively. The red-hatched lines in panel A demark two CBC cells separated by a mature Paneth cell. Blue-hatched lines in panels B-D outline borders of immature Paneth cells arising from premature differentiation of CBC cells. Note that differentiating CBC cells also undergo hypertrophy. Scale bar: 10 µm (A, B, D), 5 µm (C, E).</p