Schematic Diagram of the Protocol Used by Aboody and Colleagues From [6].

<p>Schematic Diagram of the Protocol Used by Aboody and Colleagues From [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030482#pmed-0030482-b006" target="_blank">6</a>].</p

Proliferative response of LRCs upon radiation-induced tissue injury.

<p>(A) Experimental scheme. (b–d) IF analysis for GFP (green, LRCs) and lysozyme (red, Paneth cells) relative to a non-irradiated control animal at four weeks of chase (B) and to two irradiated animals that were treated according to the scheme in (A) analyzed at 32 hours after irradiation (C,D). (E) Overlay of representative image of FACS analysis of intestinal epithelial cells of non-irradiated animals at four weeks of chase (black) compared to animals at 32 hours after whole-body irradiation (red, treated according to the scheme in 6A) for H2B-GFP intensity. Two GFP-positive populations, GFP^hi and GFP^med are resolved. Following radiation-induced tissue injury, a new GFP^med population appears (red peak), which is almost absent in untreated control animals (black line). (F) BrdU uptake as a measure of the LRCs’ proliferative response to tissue injury. The GFP^hi and GFP^med populations were evaluated for the percentage of BrdU⁺ cells at 28 and 32 hrs following radiation as depicted in A. The average BrdU levels (±s.d.) measured in 2 independent mice are displayed. (G) CyclinD1-expression as a measure of the LRCs’ proliferative response to tissue injury. Displayed is the percentage of CyclinD1-expressing cells from all Actb-positive ones as determined by single-cell RT-PCR. LRCs were obtained from three pulse chased animals; GFP^hi and GFP^med cells were obtained from three pulse-chased animals and isolated 36 hrs. after irradiation. Single cell RT-PCR was carried out on a total of 3×90 LRCs, 3×60 GFP^hi and 3×120 GFP^med cells. Single cells were only included in the analysis when the housekeeping gene Actb was expressed. The average percentages of CyclinD1-expressing cells from all Actb-positive ones (±s.d.) and the corresponding p-values obtained by two-sample t-test (* <0.02) are shown. (h) Percentage of GFP^hi and GFP^med cells expressing Defa1, Bmi1, and Lgr5 as determined by single cell RT-PCR. FACSorted GFP^hi and GFP^med cells were isolated 36 hrs. after radiation-induced tissue injury. Single-cell RT-PCR was carried out on a total of 2×30 GFP^hi and 2×60 GFP^med cells from each one male and one female mouse. Gene expression status of single cells was only included in the charts when the housekeeping gene Actb was expressed. The average proportion of cells expressing each gene (±s.d.) is displayed.</p

Organoid-formation assay.

<p>(A) Organoid-forming capacity of sorted populations from villin-rtTA/TRE-H2B-GFP mice, chased for 72–78 days. Displayed are average percentages of organoids formed (±s.e.m.) from 5000 FACS-sorted and plated events in 5 independent experiments as scored 14 days after plating. (B–C) To assess the presence of doublets among the sorted LRCs, the different subpopulations were examined by confocal microscopy. 500 cells were sorted in each well of a 6-well plate, imaged by confocal microscopy (B) and counted (C). (B) Representative IF images showing the composition of the LRC CD24^hiSSC^hi population (left) and the H2B-GFP^- CD24^hiSSC^hi population (right). (C) Quantification of the composition of FACS-sorted LRC CD24^hiSSC^hi (left) and H2B-GFP^- CD24^hiSSC^hi (right) by morphology for single cells and aggregates of differently sized cells; displayed are percentages.</p

Characterization of LRCs for Paneth and stem cell markers.

<p>(A) Co-staining of small intestinal sections from villin-rtTA/TRE-H2B-GFP mice chased for 79 days of LRCs (arrow and arrowhead, GFP, green) and Paneth cells (lysozyme; red). Several (arrow) but not all (arrowhead) LRCs are positive for the Paneth cell marker lysozyme. Imaginary cell boundaries have been drawn (white dotted line). The number of lysozyme-positive and lysozyme-negative LRCs was quantified at chase day 41 and 70. Between 29% (chase 41 days) and 39.9% of LRCs are not expressing lysozyme. (B) Representative FACS image of mouse small intestinal epithelial crypt cells analyzed for the Paneth cell marker CD24. Paneth cells fall commonly within the CD24^hiSSC^hi gate. The size of each population is indicated as percentage of live epithelial cells. (C) Representative FACS image of label retaining cells (chase 75 days) analyzed for the Paneth cell marker CD24. The size of each population is indicated as percentage of live epithelial cells. The majority of LRCs falls directly into the CD24^hiSSC^hi gate, while others scatter just around the CD24^hiSSC^hi gate in close proximity. Some of these cells in close proximity to the CD24^hiSSC^hi gate fall within the upper edge of the CD24^med gate. Of note, only a very small minority of LRCs falls outside of this cell cluster and is CD24^- (5.54%). (D) Composition of LRCs according to CD24. Displayed are averages of five independent mice chase for 72 to 78 days (± s.d.). The percentage of CD24 subpopulations within the LRCs does not add up to 100%, as some cells fall outside of the drawn gates. (E) Plots of relative gene expression levels (qRT-PCR) of Defa1, Lgr5, Bmi1, Msi1, Tert, Dll1, Lrig1, and Prox1. CD24^hiSSC^hi LRCs (dark green) and their H2B-GFP-negative counterpart (grey) were sorted from five different animals and analyzed by qRT-PCR. Comparison of the marker expression at the two different chase time points (64 days and 83 days) did not show any significant differences (data not shown). Therefore, data from all five animals were analyzed together and shown here. Displayed are averaged β-actin normalized values (± s.e.m.) and the corresponding p-values obtained by two-sample t-test (* <0.02; ** <0.005; *** <2x10e-6). Analysis of Lgr5 was also performed in two populations sorted from 3 Lgr5-EGFP mice, namely Lgr5⁺ (blue) cells and Lgr5^-CD24^hiSSC^hi (black) cells, to compare the Lgr5 expression levels in Lgr5-EGFP cells (blue) with that of CD24^hiSSC^hi LRCs (dark green).</p

Analysis of proliferation and differentiation markers in LRCs.

<p>(A–B) Progressive loss of expression of differentiation and proliferation markers in LRCs following doxycycline withdrawal. The enteroendocrine differentiation marker synaptophysin (A) and the proliferation marker Ki-67 (B) were detected by APC-A whereas FITC-A detects H2B-GFP. The size of each population is indicated as the percentage of epithelial cells. (C–D) Analysis of small intestinal sections from villin-rtTA/TRE-H2B-GFP mice chased for 5 weeks. (C) LRCs (GFP, green) and cycling cells (ki-67, red), the white arrowhead points to a cycling crypt base columnar cell (ki-67, red); (D) LRCs (GFP, arrowheads, brown) and goblet cell marker PAS (arrows, red).</p

Overview of the phenotypic comparisons between <i>Apc</i>^+/1572T and other <i>Apc</i>-mutant mouse models.

<p>Notes: Incidence is given as percentage of affected animals.</p>1,3<p>Animals were sacrificed when signs of discomfort were apparent and/or when tumor size reached 2 cm.</p>2<p>The incidence of GI tumors was calculated after exclusion of the pyloric lesions as these present in clusters often difficult to count.</p>3<p>The multiplicity of GI tumors was calculated based on all animals with the exception of those where the high tumor burden made the count not feasible.</p>*<p>This specific animal was found to carry a single tumor at 21 months of age, likely to represent a sporadic case. Background (B) of the different strains analyzed: F1: C57Bl6/J x 129Ola; Ola: inbred 129Ola; B6: inbred C57Bl6/J. n.d. not determined.</p

Biochemical characterization of the targeted <i>Apc</i>1572T allele.

<p>(A) Schematic representation of the APC tumor suppressor protein, its functional domains, and the truncated proteins resulting from the <i>Apc</i>1572T, <i>Apc</i>1638N, and <i>Apc</i>1638T targeted alleles. Only residual amounts (2%) of the truncated Apc1638N protein are encoded by the targeted allele, as shown by immuno-precipitation analysis of <i>Apc</i>^1638N/1638N ES lines <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000547#pgen.1000547-Kielman1" target="_blank">[5]</a>. (B) β-catenin/TCF reporter assay (TOP-FLASH) analysis of <i>Apc</i>^+/+ (1) and <i>Apc</i>-mutant ES cell lines: Apc^1638T/1638T (2); Apc^1572T/1572T (3); Apc^1638N/1638N (4). Each bar represents the average measurement of the luciferase units from triplicate assays. For each cell line, 3 independent experiments were performed with the TOP (filled bars) and FOP (empty bars) reporter constructs. The bold figures represent the average TOP/FOP ratio of all independent experiments. Depicted error bars correspond to standard deviation. In brief, ES cells were plated on dishes coated with MEFs and subsequently transfected by lipofection with either the TOP-FLASH or FOP-FLASH reporter constructs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000547#pgen.1000547-Korinek1" target="_blank">[10]</a> together with the Renilla luciferase vector for normalization purposes. (C) Immuno-precipitation (IP) analysis of Apc-bound β-catenin in <i>Ap</i>c-mutant ES cell lines. For comparative purposes, immuno-precipitates obtained from equal amounts of total cellular lysates were loaded.</p

Phenotypic and molecular analysis of the compound <i>in cis Apc</i>^+/1572T/<i>Smad4</i>^+/Sad mouse model.

<p>(A) Schematic illustration of the chr. 18 LOH event in intestinal tumors from <i>in cis Apc</i>^+/1572T/<i>Smad4</i>^+/Sad mice leading to loss of both <i>Smad4</i> and <i>Apc</i> wild-type alleles. (B) Comparative phenotypic analysis of the intestinal and mammary tumor predisposition among <i>Apc</i>^+/1572T, <i>Smad4</i>^+/Sad, and <i>Apc</i>^+/1572T/<i>Smad4</i>^+/Sad mice. <i>Notes</i>: (1) The incidence of GI tumors was calculated after exclusion of the pyloric lesions as these present in clusters often difficult to count. (2) The multiplicity of GI tumors was calculated based on all animals with the exception of those where the high tumor burden made the count not feasible. The asterisks indicate that the apparent absence of intestinal tumor in <i>Smad4</i>^+/Sad control animals is not in contradiction with what previously published. These mice were sacrificed at time points matched with the ages at which compound <i>Apc</i>^+/1572T/<i>Smad4</i>^+/Sad mice had to be sacrificed due to the high GI and mammary tumor burden (♀: 90.4 days +/−28.4; ♂: 118.5 days +/−26.2). However, in <i>Smad4</i>^+/Sad animals the majority of the tumors appear at 9 months of age <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000547#pgen.1000547-Alberici1" target="_blank">[15]</a>. (C) H&E staining of intestinal tumor sections from <i>Apc</i>^+/1572T (top), <i>Apc</i>^+/1572T/<i>Smad4</i>^+/Sad (middle), and <i>Smad4</i>^+/Sad (bottom) mice. (D) Smad4 IHC analysis of two intestinal adenomas from <i>Apc</i>^+/1572T/<i>Smad4</i>^+/Sad mice showing loss of Smad4 expression. LOH was observed in 100% of the polyps (n = 15) analyzed. PCR–based LOH analysis of the same cohort of <i>Apc</i>^+/1572T/<i>Smad4</i>^+/Sad polyps revealed loss of wild-type <i>Apc</i> allele in 87% of the cases (13/15; data not shown).</p

Differentiation.

<p>HE staining of mammary tumors (A–B) and pulmonary metastases (C) from <i>Apc</i>^+/1572T mice shows typical mammary glandular architecture and squamous differentiation. (D–F) Luminal epithelial differentiation as shown by cytokeratin 8 (Ck8) IHC staining. (G–I) Myoepithelial differentiation revealed by IHC staining with the Sma antibody. (J–L) IHC analysis with antibodies directed against cytokeratin 14 (Ck14) confirm the presence of squamous differentiation (hair follicle and skin cellular types). (M–O) β-catenin IHC analysis shows heterogeneous subcellular localization and intracellular accumulation with fewer cells characterized by positive nuclear staining. The results shown in this figure were confirmed in 12 independent primary tumors.</p

Phenotypic characterization of <i>Apc</i>^+/1572T mice: mammary adenocarcinomas are composed by mixed differentiation lineages with heterogeneous patterns of β-catenin intracellular accumulation and subcellular localization.

<p>Survival curves of (A) female and (B) male <i>Apc</i>^+/1572T mice, respectively. The black, green and red lines are representative of mice in the 129Ola, F1 B6x129Ola, and B6 respectively. Please note that in these graphs, age of death represents the moment at which, due to the presence of signs of discomfort or because the tumor size exceeded 2 cm³, mice had to be euthanized according to institutional and national regulations. (C) Macroscopic image of the appearance of the mammary adenocarcinomas characteristic of the <i>Apc</i>1572T model. (D) Examples of global digital microscopy scans of two mammary adenocarcinomas from <i>Apc</i>^+/1572T mice illustrative of the multi-lineage nature of these lesions.</p