Adenomatous polyposis coli (APC) is required for normal development of skin and thymus

Abstract

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.

Figure 1. Generation of the Conditional Apc Allele

(A) Schematic diagram of exons 14 and 15 of the mouse Apc 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. (B) Southern blot analysis of NdeI-digested genomic tail DNA isolated from F1 mice of various Apc mouse lines (Apc^CKON, Apc^Δ580), hybridized to a 600-bp probe. Tail genomic DNA from Apc^CKON F1 mice derived from a modified ES clone showed a 12-kb band for the Apc^CKON allele and a 10-kb band for the wild-type allele, whereas genomic DNA from the Apc^Δ580 mouse was heterozygous for the Apc^Δ580 allele (9.2-kb band). (C) Kaplan-Meier survival plot of Apc^CKO/+ mice (thin solid line, n = 39), Apc^CKO/CKO mice (thin dotted line, n = 57), Apc^Δ580/+ mice (solid line, n = 51), and wild-type littermates (broken line, n = 21). Heterozygosity of the Apc^Δ580 allele led to a significantly shortened survival (p < 0.0001), whereas those of heterozygous and homozygous Apc^CKO mice had no significant difference to that of wild-type littermates.

Figure 2. Postnatal Mortality and Stunted Growth in K14-cre; Apc^CKO/CKO Mutant Mice

Animals whose genotype is either heterozygous or homozygous for the wild-type Apc allele are referred to as normal (N); those whose genotype are K14-cre; Apc^CKO/CKO and show the presence of K14-cre–recombined mutant Apc allele are called mutant (M). (A) Two P3 mutant mice, M1 and M2, and their normal littermates, showing size variation among mutants. (B) P8 mutant mouse (right) and a normal littermate. Note sparseness of hair coat and abnormal ears. (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. (E) A P17 mutant mouse (right) with its littermate. Its bare forehead, dorsal median line, and abnormal ears are evident. (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 (p < 0.05). (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. (H) Skeletal preparations of normal (left) and mutant (right), showing differences in development of both incisor (I) and molar (M) teeth.

Figure 3. Tissue-Specific Detection and Expression of Deleted Apc Alleles

(A) Tissue-specific genotyping PCR. Only genomic DNA samples from the skin (S) and thymus (T), but not liver (L) of mice positive for K14-cre show the presence of deleted Apc^Δ580 allele. (B) Genotype- and tissue-specific expression of the truncated Apc 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 K14-cre have transcripts from both wild-type (528 bp) and deleted (313 bp) Apc alleles.

Figure 4. Histological and Immunochemical Examination of P12 Skin and Teeth

(A–E) P12 normal skin. (F–J) P12 mutant skin. (K–N) P12 normal oral cavity. (O–R) P12 mutant oral cavity. Stained with H&E for histology (A, F, K–L, O–P), Ki67 (B, G), β-catenin (C, H, M, Q), K14 (D, I, N, R), and K6 (E, J). Aberrant follicular morphogenesis, characterized by formation of irregularly spaced, nonpolarized hair follicles, in mutant skin is evident. Despite the abnormal histology, proliferation seems to be confined to hair bulb-like structures (arrows in [G], inset [G′] at higher magnification), but in mutant skin (arrows in [H], inset [H′] at higher magnification) and oral cavity (arrows in insets [Q′] at higher magnification) elevated cytosolic localization of β-catenin is detected in some cells. Scale bars: 50 μm for (A–F), (H–J); 250 μm for (K) and (O); 100 μm for (G), (L–N), (P–R); 20 μm for (Q′).

Figure 5. Expression of Shh and β-catenin Transcripts in Normal (Apc^CKO/CKO) and Mutant (K14-cre; Apc^CKO/CKO) Embryonic Skin

(A–D) Section ISH with Shh 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 K14-driven Apc loss caused aberrant pattern formation (F′) and formed ectopic hair placodes in normally hairless foot pads (H, arrows) which are absent in normal (G).

Figure 6. Histological and Immunochemical Examination of Thymus

(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.