K14-Driven Loss of Apc Results in Severe Growth Retardation and Early Lethality
To introduce the mutation of Apc into cells expressing K14, we crossed WW6 ES cell–derived [15] ApcCKO/+ mice with K14-cre recombinase mice in FVB background [16]. The K14-cre; ApcCKO/+ mice were normal in appearance and were fertile. K14-cre; ApcCKO/+ males were crossed to ApcCKO/CKO females to avoid the potential deleter effect in oocytes of K14-cre–positive females [17]. The mice were intercrossed thereafter for maintenance; hence, the mice used for analysis were in a mixed background of FVB, 129/S, and C57BL/6 in similar proportions, with minimal contribution of SJ.
The K14 promoter is a commonly used epidermal cell promoter because of its expression by the mitotically active cells of the epidermis and its appendages in mature skin [18], but most notably it is active in embryonic ectoderm as early as the single layered ectodermal stage of embryonic day (E) 9.5 [19]. A restricted expression of K14 is also found in thymic epithelial cells (TECs) in the medulla of normal thymus [20].
We genotyped a total of 458 pups (8–10 d old) from 67 litters resulting from crosses between K14-cre; ApcCKO/+ and ApcCKO/CKO mice. The mutant mice of the genotype K14-cre; ApcCKO/CKO (hereafter, KA mutant) were born, but the observed frequency of KA mutants was much less than expected (78 of 458 [17.0%]; p < 0.0005 Chi-square analysis, Table 1). To assess the basis for the neonatal lethality of KA mutants, we monitored three litters from birth to weaning by measuring the body weight of each pup every day. A total of 25 pups were born from three litters, of which 7 (28%) were confirmed to be K14-cre; ApcCKO/CKO by genotyping, indicating that KA mutants were born in the expected Mendelian ratio. The KA mutant pups were nursed normally, and there was milk in their stomachs during the first 2 or 3 d after birth, but they failed to thrive (Figure 2). By postnatal day (P) 8–10, at the time of genotyping, many KA mutant pups were considerably smaller than their littermates (Figure 2B–2F) and some have died at or prior to this age. None of KA mutants survived to weaning age.
Table 1  Genotype Distribution of Progeny from the Matings
Figure 2  Postnatal Mortality and Stunted Growth in K14-cre; ApcCKO/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; ApcCKO/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. The ability of whole embryos to exclude blue dye was used to examine the epidermal barrier, normally acquired beginning at E16 and complete by E18.5 [21]. Analyses of E17.5–E18.5 KA mutants showed that they were able to exclude blue dye, indicating that the epidermal barrier was intact (Figure S2). At these embryonic ages, there were no differences in size between the mutants and their littermates, but the mutants showed a patch of “birthmark” or dark pigmentation on their foreheads and a dark median line that ran caudally from head to tail. Their external ears or pinnae were shriveled in appearance and pigmented compared to those of littermates.
External characteristics of KA mutants that were evident at E18.5 persisted after birth and became more prominent as they grew (Figure 2A–2F). Growth of pelage hair was generally delayed in the mutants. At around P8, the KA mutants were hairless and had wrinkled skin while their phenotypically normal littermates had a smooth thin coat of hair (Figure 2B). At this age, two lower incisors start to erupt in normal littermates and these were absent in the KA mutants (Figure 2C and 2D). Animals also tended to be smaller and around P10–P12 displayed abnormally short and misshapen vibrissae and short, shaggy pelage hairs (Figure 2C and 2D). Development of thick ridges in their skin, particularly around the ears, eyelids, forehead, nose, and paws, became noticeable (Figure 2E). These regions looked scaly, and these animals hardly kept their eyes open. In contrast to the normal littermates that consistently increased their body weight with age, surviving KA mutants started to lose weight from P10 onwards; by P16–P17 they were all lethargic, and none of them survived to weaning (Figure 2E and 2F). At the time of autopsy all the mutants were toothless, without incisors or molars, and their stomachs were consistently small and had no solid food, unlike their age-matched littermates, suggesting that the observed weight loss could be the result of failure to ingest solid food (Figure 2F). Interestingly, changes in body weights and timing of hair growth varied considerably among mutant pups even if they were from the same litter, whereas those of phenotypically normal littermates tended to be similar. This difference was also reflected in the variation in timing of death in mutants: some mutant pups were born alive but died within a day or two, some survived close to the weaning age. This variability of the mutant phenotypes suggests possible variation in the timing and efficiency of cre-mediated Apc deletion. It is possible that the genetic background has a role to play in this variability.
Gross examination of internal organs also showed that the mutants' thymi were consistently inconspicuous and were very small for their age, whereas those of their littermates were very prominent in size (Figure 2G). This difference was evident as early as P3. Quite frequently mutant thymi in P12–P17 mutant mice also contained black deposits within the tissue (unpublished data). Mutant mice were also examined for any skeletal abnormalities by preparing skeletal specimens of P16–P17 mice stained with Alizarin red. No differences between the normal and KA mutant mice in the mandibular bone can be detected, but the mutant mice lacked or had underdeveloped set of maxillary incisors and molars (Figure 2H). We detected no other major skeletal abnormalities.