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

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

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

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

Teratoma formation assays indicate an intermediate differentiation defect in <i>Apc</i>^1572T/1572T ES cells.

<p>(A) HE analysis of normal mammary gland with luminal cells surrounded by a basal layer of myoepithelial cells. (B) IF analysis of normal mammary glands for Ck8 (luminal cells, green) and Sma (myoepithelial, red). (C) HE staining of <i>Apc</i>^1572T/1572T teratoma showing the typical mammary gland architecture with lobular and ductal structures. (D) IF analysis of <i>Apc</i>^1572T/1572T teratomas for luminal and myoepithelial cell types. The frequency of these structures in teratomas derived from <i>Apc</i>^1572T/1572T ES cells is largely increased when compared with (F) teratomas derived from <i>Apc</i>^+/+ ES cells. (E) Summary of the results of the teratoma differentiation assays of <i>Apc</i>-mutant ES cells. Antibodies employed to evaluate differentiation are: Glial Fibrillary Acidic Protein (GFAP) for glial cells; 2H3 for neurofilaments; SV2 for synaptic vesicles; A4.1025 for adult myosin. Mammary gland structures were primarily identified by HE and then confirmed by IF as shown in panels a–d; also in the case of cartilage and epithelial structures HE stained section were employed. n.d. not determined. Differentiation levels were scored as: (−) not present; (−*) vestigial presence; (+) present; (++) highly abundant. The shaded areas indicate groups of teratomas for which the corresponding antibody staining was negative.</p

LOH analysis of <i>Apc</i>^+/1572T mammary adenocarcinomas.

<p>(A) PCR–based LOH analysis of tumour DNA samples amplified in the presence of radioactive nucleotides as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000547#pgen.1000547-Smits3" target="_blank">[35]</a>. Samples were scored as having lost the wild type allele when the ratio between the intensity of the two alleles was ≤0.6. <i>Apc</i>^+/1572T mammary tumor samples (T); <i>Apc</i>^+/+ DNA control (C₁); <i>Apc</i>^+/1572T DNA control (C₂). Out of the 27 samples (collected from 23 tumours) analysed, only two (lanes marked by an asterisks) show a ratio higher than 0.6 and were accordingly scored as not having allelic imbalance. (B) Western analysis of tumour-derived total protein lysates confirms the somatic loss of full length Apc. Tumour samples (T); wild type control (C₃); <i>Apc</i>^+/1572T control from tail sample (C₄).</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

Rescue of <i>Tcf3</i> expression in <i>Apc</i>NN ESCs partially restores <i>in vivo</i> neural differentiation.

<p>Teratoma samples were obtained from wild type, <i>Apc</i>NN and <i>Apc</i>NN stably expressing <i>Tcf3</i> (Tcf3 OE) ESCs. Tissue sections were stained by H&E, thionin (marker of cartilage differentiation), and by IHC with specific antibodies against the neural differentiation markers GFAP, 2H3 (neurofilaments) and SV2 (synaptic vesicles). Oct4 IHC analysis was used to asses the presence of undifferentiated EC-like cells in the teratomas.</p

Wnt signaling downregulates Tcf3 expression in mouse ESCs.

<p>A. qRT-PCR analysis of <i>Tcf3</i> in wild type, <i>Apc</i>NN and <i>Apc</i>^Min/Min ESCs. <i>Actb</i> was used as an internal control; bars represent n = 2 ± SD. B. Western blot analysis of the core pluripotency markers Oct4, Nanog, Sox2 and Tcf3 on protein lysates isolated from two independent <i>Apc</i>NN clones and wild type control ESCs. Actb and Tubulin were used as an internal control. C–D. qRT-PCR analysis of <i>Tcf3</i> in wild type ESCs treated for different time intervals with Wnt3a conditioned medium (C), or with the GSK-inhibitor SB-216763 (D). L-medium and DMSO were employed as controls, respectively. <i>Actb</i> was used as an internal control; bars represent n = 2 ± SD. E. Time course western blot analysis of Tcf3 expression in wild type ESCs treated with Wnt3a conditioned medium (Wnt3a CM) or control L-medium (LM). <i>Actb</i> was used as an internal control. F. qRT-PCR analysis of <i>Tcf3</i> and Wnt target genes Axin2 and Cdx1 in wild type ESC treated for 48 h with different concentrations of GSK-inhibitor SB-216763 or DMSO as control. <i>Actb</i> was used as an internal control; bars represent n = 2 ± SD.</p

The Wnt-regulated miR-211 targets Tcf3 in mouse ESCs.

<p>A. qRT-PCR analysis showing a dosage-dependent up-regulation of miR-211 in different <i>Apc</i>-mutant ESCs. SnoRNA-234 was used as an internal control; bars represent n = 2±SD. B–C. Time course analysis of wild type ESCs treated with Wnt3a conditioned medium (B) or with the GSK-inhibitor SB-216763 (C). L-medium and DMSO were used as controls, respectively. RNAs were isolated at different time points and were subjected to qRT-PCR analysis of miR-211 or snoRNA-234 as an internal control. Bars represent n = 2±SD. D. Western blot analysis of Tcf3 expression in protein lysates isolated from independent clones of wild type ESCs stably expressing miR-211 (miR-211 OE) or the corresponding empty vector (control). Two independent <i>Apc</i>NN clones were included for comparison. E. Schematic representation of the Tcf3-3′-UTR luciferase vector derived from the pmirGLO construct (Promega). Sequence alignment between miR-211 and its target site on Tcf3-3′-UTR. Site directed mutagenesis was used to introduce 7-bp or 4-bp mutations in Tcf3-3′-UTR. F. HEK-293 cells were co-transfected with the Tcf3-3′-UTR luciferase vector, and either with miR-211 or a non-targeting miRNA. Luciferase activity was measured 24 h post-transfection and normalized to Renilla luciferase signal. The same experiment was repeated with the mutant luciferase vectors, MTR1 and MTR2. Asterix represent <i>P</i>-value<0.01 and bars represent n = 3±SEM. G. Flow cytometric analysis of Tuj1 and Nestin in miR-211 over expressing ESCs (miR-211 OE) and their controls (Emp) after 13 days of neural differentiation. Two independent clones were used for each genotype and representative example of each genotype is shown. Numbers in the graph represent the percent of cells in neural (green), progenitor (red) or negative (blue) populations. H. Histogram showing the relative expression of early neural markers <i>Fgf5</i>, <i>Nestin</i>, <i>Pax6</i> and <i>Sfrp2</i> in embryoid bodies derived from independent wild type ESCs clones stably expressing miR-211 or the corresponding empty vector. RNAs were isolated at different time points and were analyzed by qRT-PCR for different lineage markers. Bars represent n = 2±SD. I. qRT-PCR analysis of <i>Fgf5</i>, <i>Nestin</i>, <i>Pax6</i> and <i>Sfrp2</i> in wild type ESCs stably expressing miR-211 or the corresponding empty vector, cultured for 24 h in N2B27 medium. Bars represent n = 2±SD.</p