Generation of Pref-1-null mice.
To determine the role of Pref-1 in growth and adipogenesis and the contribution of Pref-1 in mouse mUPD12 and human mUPD14 syndromes, we used gene targeting to generate Pref-1-null mice. The genomic pref-1
allele was disrupted by insertion of a neomycin resistance cassette into exons 2 and 3 of pref-1
(Fig. ). G418- and ganciclovir-resistant clones were screened for homologous recombination, and the positive clones were microinjected into 3.5-day C57BL/6J or BALB/c blastocysts to generate chimeric animals. Germ line transmission from each clone was obtained, and no subsequent difference between separate lines was observed. Male chimeras were mated with either C57BL/6J or BALB/c females to produce Pref-1 heterozygotes (+/−). Intercrosses of Pref-1+/−
mice produced Pref-1-null mice. The wild-type (7.0 or 5.5 kb), heterozygote (7.0 and 5.2 kb or 7.3 and 5.5 kb), or homozygote (5.2 or 7.3 kb) Pref-1 alleles were detected from F2
pups generated by intermating F1
heterozygotes (Fig. B). To confirm the absence of normal Pref-1 protein in Pref-1-null embryos (E17.5), we performed Western blot analysis with polyclonal antibodies against murine Pref-1 that we had prepared by immunization of rabbits with bacterially expressed murine Pref-1 (25
). Figure shows that in wild-type embryos, Pref-1 antibody detected the multiple forms of Pref-1 protein, which we have previously demonstrated were derived from alternative splicing and posttranslational modification (25
). Pref-1 protein was not detected in homozygous embryos.
FIG. 1. Targeted disruption of the pref-1 gene. (A) The top diagram is a schematic representation of the wild-type pref-1 allele with five exons. Pref-1 exons are represented by black boxes. The middle diagram shows the targeting construct with pPNT-NEO and thymidine (more ...)
Mice lacking Pref-1 displayed pre- and postnatal growth retardation, eyelid, and skeletal abnormalities. The average litter size of Pref-1-null mice was significantly lower (4.26 ± 0.26 pups per litter, n
= 25) than those of wild-type mice (6.75 ± 0.32 pups per litter, n
= 27, P
< 0.001), suggesting that Pref-1 plays an important function in embryonic development. We also measured the embryo size at E18.5 of mice generated by interbreeding of Pref-1 heterozygotes. Pref-1-null embryos (0.96 ± 0.004 g, n
= 9) at E18.5 were smaller than wild-type littermates (1.18 ± 0.003 g, n
= 12, P
< 0.0001) (Fig. ). Approximately 50% of null mice were dead within 2 days of birth, indicating that Pref-1 is important for perinatal growth and survival. We observed a similar degree of neonatal mortality in litters produced from the mating of wild-type females and Pref-1-null or heterozygous male mice, which, as described below, do not express Pref-1. This suggests that the survival rate is entirely dependent on the phenotype of the pup rather than that of the mother. We also found skeletal malformation, including asymmetrical junction of ribs to sternum and fusion of ribs from embryos and postnatal dead mice (Fig. ). Skeletal abnormalities and pulmonary defects, as well as other problems arising from these defects, such as poor suckling ability, may have contributed to the high rate of postnatal death observed in null mice (8
). As shown in Fig. , Pref-1-null mice also showed eyelid abnormalities similar to the blepharophimosis observed in individuals with a deletion at 14q32 compassing the pref-1
locus or in mUPD14 patients (29
). However, this was strain dependent and was observed in Pref-1-null mice on a BALB/cJ background, but not on a C57BL/6J background.
FIG. 2. Pre- and postnatal growth retardation and eyelid abnormalities in mice lacking Pref-1. (A) Intrauterine growth retardation. Pref-1-null embryos are smaller than wild-type embryos at E18.5. The embryos were generated from crossbreeding Pref-1 heterozygotes (more ...)
At weaning, both female and male Pref-1-null mice weighed less than wild-type mice (female, 7.34 ± 0.21 g, n = 32, versus 8.51 ± 0.19 g, n = 27, P < 0.0001; male, 7.58 ± 0.25 g, n = 36, versus 8.81 ± 0.21 g, n = 37, P < 0.0001, respectively). Initially, after weaning at 3 weeks of age, the mice were fed a regular chow diet. At 15 weeks of age, we found that there was growth retardation with moderately larger fat pad mass in Pref-1-null mice (data not shown). In an attempt to observe the Pref-1 effect on adipose tissue development more clearly, we carried out all subsequent studies with the mice that were fed a high-fat (45 kcal%) diet as described in Materials and Methods. Pref-1-null mice on a high-fat diet gained weight at a slower rate during the immediate postweaning period. However, as shown in Fig. , there was a catching up in body weight at approximately 90 days for females and 65 days for males. By 16 weeks of age, the body weights were not different between null and wild-type mice (female, 24.94 ± 0.99 g, n = 14, versus 25.35 ± 0.50 g, n = 11; male, 35.60 ± 0.97 g, n = 14, versus 35.72 ± 0.37 g, n = 16). These data suggest to us that accelerated weight gain at this later stage could be due to increases in adipose tissue mass.
Increased adiposity in mice lacking Pref-1.
We investigated whether the accelerated weight gain of null mice observed at later stages is due to fat accretion. At an early age (8 weeks), the white fat pad weights including inguinal, retroperitoneal, and reproductive fats were similar between null mice and wild-type mice (female, 3.80% ± 0.37%, n = 7, versus 3.33% ± 0.62%, n = 10, P = 0.2061; male, 3.17% ± 0.33%, n = 7, versus 3.12% ± 0.34%, n = 4, P = 0.9286). However, as shown in Fig. , at 16 weeks of age, the white fat pads (expressed as a percentage of body weight) from female Pref-1-null mice after 13 weeks of feeding of a high-fat diet (45 kcal%) weighed significantly more than those of wild-type mice (inguinal, 2.34% ± 0.2% versus 1.64% ± 0.1%, P = 0.0182; retroperitoneal, 1.50% ± 0.1% versus 0.90% ± 0.1%, P = 0.0096; parametrial, 3.16% ± 0.5% versus 1.78% ± 0.2%, P = 0.0296; and brown fat, 0.29% ± 0.01% versus 0.22% ± 0.01%, P = 0.0045) for null (n = 10) versus wild-type (n = 7) mice, respectively. Figure also shows that similar increases in fat pad mass were observed in all regions of male Pref-1-null mice, except the epididymal fat pad. In contrast to fat pad weights, the weights of other organs, including lungs and kidneys, of the Pref-1-null mice at later ages did not catch up but remained smaller than those of wild-type mice. Interestingly, as summarized in Table , Pref-1-null mice developed enlarged livers (female, 4.41% ± 0.13%, n = 9, versus 3.77% ± 0.08%, n = 7, P = 0.001; male, 4.10% ± 0.14%, n = 7, versus 3.60% ± 0.12%, n = 7, P = 0.0224) with increased total lipid content (female, 8.77 ± 0.57 mg/g of liver, n = 4, versus 5.76 ± 0.31 mg/g of liver, n = 7, P = 0.0046; male, 10.73 ± 0.69 mg/g of liver, n = 5, versus 8.35 ± 0.44 mg/g of liver, n = 5, P = 0.0239).
FIG. 3. Accelerated adiposity in mice lacking Pref-1. (A) Percentage of fat pad and organ weight relative to body weight. Fat depots and organs were dissected from 16-week-old wild-type (WT) and Pref-1-null mice fed a high-fat diet (n = 7 to 10 per group). (more ...)
Litter size, embryo and body weights, fat pad weights, lipid metabolites, and total lipid contents in livers of wild-type and Pref-1-null micea
We next determined whether increased fat mass in Pref-1-null mice results from cellular hypertrophy or hyperplasia. Even though the fat pad weight of null mice was higher than that of wild-type mice, total DNA contents, indicative of total cell number in adipose tissue that includes both preadipocytes and adipocytes, did not differ (Table ). As shown in Fig. , staining of paraffin sections and examination of the cell size distribution of inguinal fat pads from 16-week-old female mice revealed increased adipocyte cell size, indicative of hypertrophy, in Pref-1-null compared to wild-type mice. Furthermore, mRNA levels for late markers of adipocyte differentiation, FAS, and SCD-1, were increased in Pref-1-null mice compared to wild-type mice, demonstrating enhanced adipocyte differentiation (Fig. ). We also found that the enlarged fatty liver of Pref-1-null mice showed the increase in FAS and SCD-1 expression that normally accompanies increased hepatic lipogenesis and fat synthesis.
Pref-1 is expressed only from the paternal allele.
Because Pref-1 is expressed only from the paternal allele, heterozygotes with either maternal or paternal inheritance of the pref-1 knockout allele (m−/+ or +/p−) should have phenotypes similar to those of wild-type or null mice, respectively. To specifically address this, male or female pref-1 heterozygotes (+/−) were mated with wild-type mice. Western blot analysis of the E17.5 embryo in Fig. shows that Pref-1 protein expression was completely abolished in heterozygotes with the paternally-inherited pref-1 knockout allele (+/p−). On the other hand, heterozygotes with the maternally inherited pref-1 knockout allele (m−/+) express Pref-1 protein at a level similar to that of wild-type animals. These results clearly demonstrate that Pref-1 is expressed only from the paternal allele. The body weight of these animals (m−/+; +/p−) was examined at days 21 and 56—time points at which we had demonstrated a significant decrease in body weight for Pref-1-null mice. Figure illustrates that, as predicted, at 21 days of age, Pref-1 heterozygotes with the paternally inherited pref-1 knockout allele (+/p−) exhibited the same growth retardation that we observed for Pref-1-null mice [female, 9.13 ± 0.25 g, n = 9 (+/+) versus 6.66 ± 0.17 g, n = 5 (+/p−), P < 0.0001; male, 9.71 ± 0.43 g, n = 7 (+/+) versus 7.51 ± 0.17 g, n = 7 (+/p−), P < 0.0001]. In contrast, heterozygotes with disruption of the maternal allele (m−/+) have the same growth rate as wild-type littermates. When fat pad weight (sum of inguinal, reproductive, and perirenal fat pads) from male mice was examined after feeding on a high-fat diet from 3 to 16 weeks of age, heterozygotes with the paternally inherited pref-1 knockout allele (+/p−) displayed a 45% increase in fat pad weight compared to their wild-type littermates (m+/−, 9.45% ± 0.7%, n = 5; versus m+/+, 6.50% ± 0.7%, n = 6, P = 0.0129), the same as we observed for Pref-1-null mice (Fig. ). In contrast, heterozygotes with the maternally inherited pref-1 knockout allele (m−/+) did not show an increase. Therefore, both the early growth retardation and the increased fat mass that occurred in Pref-1-null mice were recapitulated as the result of paternal imprinting of Pref-1.
FIG. 4. Mice with maternal or paternal inheritance of the pref-1-knockout allele (m−/+ or +/p−) have phenotypes similar to those of wild-type or null mice, respectively. (A) Pref-1 protein expression in E17.5 embryos generated (more ...)
The regulation of expression of imprinting genes in the cluster that includes Pref-1 is quite complex. Gtl2 and Pref-1 have been shown to be reciprocally expressed in a manner analogous to that of the Igf2/H19
). We, therefore, examined expression of gtl2
in Pref-1-null mice and found that the expression levels for gtl2
were similar between null and wild-type mice (Fig. ). These data indicate that replacing part of the pref-1
genome (part of exons 2 and 3) with the neo
cassette to generate Pref-1-null mice, did not affect expression of the neighboring reciprocally regulated imprinted gene, gtl2
. We conclude that the phenotype we observe in Pref-1-null mice is likely attributable solely to the absence of Pref-1 expression.
FIG. 5. Expression of the gtl2 gene in wild-type and Pref-1-null mice. The expression of gtl2 was measured from whole embryo (E15.5) or white adipose tissue (WAT) (16 weeks) by RT-PCR. As reference for quantitative gene expression, cyclophilin was amplified simultaneously (more ...) Increase in lipid metabolite levels in Pref-1-null mice.
To determine whether the increased fat pad mass in Pref-1-null mice is reflected by levels of serum lipid metabolites, levels of triglycerides, free fatty acids, and cholesterol were assessed in Pref-1-null mice that were fed a high-fat diet from 3 to 16 weeks of age. Serum triglyceride was elevated substantially by 36 and 77% in Pref-1-null female and male mice, respectively (Fig. ). Pref-1-null male mice showed a 34% elevation in their free fatty acid levels in the fed state (Fig. ). Cholesterol levels in null mice were also elevated by 21% in female mice and 28% in male mice (Fig. ). The hypercholesterolemia and hyperlipidemia, as well as the enlarged, fatty liver, suggest that the loss of antiadipogenic action in Pref-1-null mice disturbs the homeostasis of lipid metabolism.
FIG. 6. Altered serum lipid metabolite levels in mice lacking Pref-1. (A, B, and C) Levels of triglycerides, free fatty acids, and cholesterol in serum were measured from Pref-1-null and wild-type (WT) mice at 16 weeks of age after feeding on a high-fat diet (more ...)