Adenomatous Polyposis Coli (APC) Is Required for Normal Development of Skin and Thymus

The tumor suppressor gene Apc (adenomatous polyposis coli) is a member of the Wnt signaling pathway that is involved in development and tumorigenesis. Heterozygous knockout mice for Apc have a tumor predisposition phenotype and homozygosity leads to embryonic lethality. To understand the role of Apc in development we generated a floxed allele. These mice were mated with a strain carrying Cre recombinase under the control of the human Keratin 14 (K14) promoter, which is active in basal cells of epidermis and other stratified epithelia. Mice homozygous for the floxed allele that also carry the K14-cre transgene were viable but had stunted growth and died before weaning. Histological and immunochemical examinations revealed that K14-cre–mediated Apc loss resulted in aberrant growth in many ectodermally derived squamous epithelia, including hair follicles, teeth, and oral and corneal epithelia. In addition, squamous metaplasia was observed in various epithelial-derived tissues, including the thymus. The aberrant growth of hair follicles and other appendages as well as the thymic abnormalities in K14-cre; Apc(CKO/CKO) mice suggest the Apc gene is crucial in embryonic cells to specify epithelial cell fates in organs that require epithelial–mesenchymal interactions for their development

Genetic Mechanisms in Apc-Mediated Mammary Tumorigenesis

Many components of Wnt/β-catenin signaling pathway also play critical roles in mammary tumor development, yet the role of the tumor suppressor gene APC (adenomatous polyposis coli) in breast oncongenesis is unclear. To better understand the role of Apc in mammary tumorigenesis, we introduced conditional Apc mutations specifically into two different mammary epithelial populations using K14-cre and WAP-cre transgenic mice that express Cre-recombinase in mammary progenitor cells and lactating luminal cells, respectively. Only the K14-cre–mediated Apc heterozygosity developed mammary adenocarcinomas demonstrating histological heterogeneity, suggesting the multilineage progenitor cell origin of these tumors. These tumors harbored truncation mutation in a defined region in the remaining wild-type allele of Apc that would retain some down-regulating activity of β-catenin signaling. Activating mutations at codons 12 and 61 of either H-Ras or K-Ras were also found in a subset of these tumors. Expression profiles of acinar-type mammary tumors from K14-cre; ApcCKO/+ mice showed luminal epithelial gene expression pattern, and clustering analysis demonstrated more correlation to MMTV-neu model than to MMTV-Wnt1. In contrast, neither WAP-cre–induced Apc heterozygous nor homozygous mutations resulted in predisposition to mammary tumorigenesis, although WAP-cre–mediated Apc deficiency resulted in severe squamous metaplasia of mammary glands. Collectively, our results suggest that not only the epithelial origin but also a certain Apc mutations are selected to achieve a specific level of β-catenin signaling optimal for mammary tumor development and explain partially the colon- but not mammary-specific tumor development in patients that carry germline mutations in APC

Expression of <i>Shh</i> and β-catenin Transcripts in Normal <i>(Apc^CKO/CKO)</i> and Mutant <i>(K14-cre; Apc^CKO/CKO)</i> Embryonic Skin

<p>(A–D) Section ISH with <i>Shh</i> probe in E14.5 normal (A), E14.5 mutant (B), E16.5 normal (C), and E16.5 mutant (D) skin. Broken lines indicate the interface between epithelium and mesenchyme. Scale bars: 50 μm. Whole mount in situ detection of β-catenin in E15.5 normal (E, G), mutant (F, H) embryos. Aberrant initiation of multiple hair placodes is evident at E14.5. Loss of <i>K14</i>-driven <i>Apc</i> loss caused aberrant pattern formation (F′) and formed ectopic hair placodes in normally hairless foot pads (H, arrows) which are absent in normal (G).</p

Postnatal Mortality and Stunted Growth in <i>K14-cre; Apc^CKO/CKO</i> Mutant Mice

<div><p>Animals whose genotype is either heterozygous or homozygous for the wild-type <i>Apc</i> allele are referred to as normal (N); those whose genotype are <i>K14-cre; Apc^CKO/CKO</i> and show the presence of <i>K14-cre</i>–recombined mutant <i>Apc</i> allele are called mutant (M).</p><p>(A) Two P3 mutant mice, M1 and M2, and their normal littermates, showing size variation among mutants.</p><p>(B) P8 mutant mouse (right) and a normal littermate. Note sparseness of hair coat and abnormal ears.</p><p>(C–D) Vibrissae of whisker pads are short and oddly angled in a P12 mutant mouse (C), relative to control (D). Note the lack of incisors in the mutant.</p><p>(E) A P17 mutant mouse (right) with its littermate. Its bare forehead, dorsal median line, and abnormal ears are evident.</p><p>(F) Growth curve of mutants and normal littermates. Mutants exhibit stunted growth, which became more prominent as they aged, and weigh significantly less than littermates from P8 (<i>p</i> < 0.05).</p><p>(G) Comparison of mutant and normal thymus from P3 mice. The mutant thymus (left) is dramatically smaller for its age compared to the normal littermate (right). The scale bar equals 1 mm.</p><p>(H) Skeletal preparations of normal (left) and mutant (right), showing differences in development of both incisor (I) and molar (M) teeth.</p></div

Tissue-Specific Detection and Expression of Deleted <i>Apc</i> Alleles

<div><p>(A) Tissue-specific genotyping PCR. Only genomic DNA samples from the skin (S) and thymus (T), but not liver (L) of mice positive for <i>K14-cre</i> show the presence of deleted <i>Apc^Δ580</i> allele.</p><p><b>(</b>B<b>)</b> Genotype- and tissue-specific expression of the truncated <i>Apc</i> transcripts. A representative gel of RT-PCR using primers F546 and R721, showing that only RNA from the skin and thymus but not liver of mice positive for <i>K14-cre</i> have transcripts from both wild-type (528 bp) and deleted (313 bp) <i>Apc</i> alleles.</p></div

Histological and Immunochemical Examination of Thymus

<p>(A–D) P3 normal thymus. (E–G) Mild P3 mutant thymus. (H–K) Severe P3 mutant thymus. (L–O) P13 mutant thymus. Stained with H&E for histology (A, E, H, L), BrdU (B, I, M), β-catenin (C, F, J, N), and K14 (D, G, K, O). (B) Actively dividing thymocytes are visible at the superficial edge of cortex of normal P3 thymus. Note the progression of histological abnormalities in the mutant thymus from mild P3, severe P3 to P13 (A, E, H, L). Scale bars, 20 μm.</p

Generation of the Conditional <i>Apc</i> Allele

<div><p>(A) Schematic diagram of exons 14 and 15 of the mouse <i>Apc</i> gene, the targeting vector, and the resulting conditional allele with 2 LoxP sites sandwiching the exon 14. The PGK-neomycin cassette was inserted within intron 14 by recombineering technique. This cassette is sandwiched by 2 FRT sites that could be removed by crossing to FLPe-expressing mice. Positions of PCR primers used for genotyping PCR (F2, R2, R4) and RT-PCR (F546 and R721) are indicated. Positions of probe used for Southern blot analysis with NdeI sites are also shown. Upon Cre-mediated recombination, exon 14 is removed and leads to truncated Apc protein, of which the first 580 aa correspond to the normal.</p><p>(B) Southern blot analysis of NdeI-digested genomic tail DNA isolated from F1 mice of various <i>Apc</i> mouse lines <i>(Apc^CKON, Apc^Δ580),</i> hybridized to a 600-bp probe. Tail genomic DNA from <i>Apc^CKON</i> F1 mice derived from a modified ES clone showed a 12-kb band for the <i>Apc^CKON</i> allele and a 10-kb band for the wild-type allele, whereas genomic DNA from the <i>Apc^Δ580</i> mouse was heterozygous for the <i>Apc^Δ580</i> allele (9.2-kb band).</p><p>(C) Kaplan-Meier survival plot of <i>Apc^CKO/+</i> mice (thin solid line, <i>n</i> = 39), <i>Apc^CKO/CKO</i> mice (thin dotted line, <i>n</i> = 57), <i>Apc^Δ580/+</i> mice (solid line, <i>n</i> = 51), and wild-type littermates (broken line, <i>n</i> = 21). Heterozygosity of the <i>Apc^Δ580</i> allele led to a significantly shortened survival (<i>p</i> < 0.0001), whereas those of heterozygous and homozygous <i>Apc^CKO</i> mice had no significant difference to that of wild-type littermates.</p></div