Rai1 duplication causes physical and behavioral phenotypes in a mouse model of dup(17)(p11.2p11.2) (original) (raw)
Correcting genomic balance normalizes Dp(11)17 phenotypic traits. We explored the possibility that a gene-dosage effect may cause the behavioral phenotypes we observed in our chromosome-engineered animal models by normalizing the gene copy numbers and then examining the resultant phenotype. Compound heterozygous Dp(11)17/Df(11)17 animals were bred. These mice contain the normal disomic copy number of each of the 19 genes within the rearrangement interval. We previously showed that the underweight phenotype observed in Dp(11)17/+ animals and the obesity observed in Df(11)17/+ animals were rescued in the Dp(11)17/Df(11)17 mice (21). Thus, normalizing the copy number of the genes within the rearranged interval normalizes the weight phenotype, which is consistent with the hypothesis that the complex phenotype in the animals with segmental aneuploidy is the result of gene dosage. To simplify the analysis of the data, all the behavioral studies were performed in male mice, since gender specificity was previously observed for Dp(11)17/+ mice (22).
We were able to rescue many of the behavioral phenotypes observed in the male animals with segmental aneuploidies by normalizing the gene copy number in Dp(11)17/Df(11)17 mice (Figures 2 and 3). The vertical activity and center/total distance ratio determined by the open-field test — used to assesses exploratory activity and providing some measure of anxiety levels, which were abnormal in Dp(11)17/+ mice — were normalized in the Dp(11)17/Df(11)17 animals (Figure 2, A and B). Surprisingly, reconstitution of the normal gene dosage in Dp(11)17/Df(11)17 animals did not normalize the total distance traveled by Dp(11)/+ mice (Figure 2C). Potential explanations for the results presented in Figure 2C include: Rai1 position effects, the presence of a _cis_-regulatory element near or within the rearranged genomic interval that regulates another gene affecting this phenotype, or limitations of the behavioral testing paradigm when evaluating relatively small numbers of animals. The percent freezing to context as determined by the conditioned fear test assessing learning and memory, which was abnormal in Dp(11)17/+ animals, was corrected when the mice were balanced genomically (Figure 3A). No significant difference was found in percent freezing to the sound cue among the different genotypes (Figure 3B).
Genomic balance restores normal behavior. The results of 3 different parameters that were measured in the open-field paradigm (vertical activity, center/total distance, and total distance) are represented as bar graphs for each of the 4 genotypes tested. (A) Vertical activity [F(3, 46) = 26.9; P < 0.0001]. (B) Center/total distance ratio [F(3, 46) = 6.740; P = 0.001]. (C) Total distance (cm) [F(3, 46) = 4.260; P = 0.01]. Note that the abnormal vertical activity and center/total distance ratio observed in Dp(11)17/+ animals was normalized after restoration of genomic balance [i.e., _Dp(11)17/Df(11)17_]. Black bars, wild type; light gray bars, Dp(11)17/+; dark gray bars, Df(11)17/+; white bars, Dp(11)17/Df(11)17. Values represent mean ± SEM. Actual values are given in Supplemental Table 2. *P < 0.05, **P < 0.01 compared with wild-type littermate.
Genomic balance corrects learning and memory deficits. The percentages of freezing to context [F(3, 46) = 10.847, P < 0.0001] (**A**) and the sound cue (_P_ > 0.05) (B) are represented for each of the 4 genotypes tested. Note that the abnormal freezing of Dp(11)17/+ animals was corrected in Dp(11)17/Df(11)17 mice. Black bars, wild type; light gray bars, Dp(11)17/+; dark gray bars, Df(11)17/+; white bars, Dp(11)17/Df(11)17. The mean ± SEM values are presented. Actual values are given in Supplemental Table 2. **P < 0.01 compared with wild type.
Correcting Rai1 copy number in Dp(11)17/+ mice. We next investigated Rai1 copy number to determine whether it was the dosage-sensitive gene responsible for the physical and behavioral phenotypes in Dp(11)17/+ mice. We have previously generated mice carrying a null mutation in Rai1 (Rai1+/– mice), and these animals manifest craniofacial abnormalities and are obese (23). By crossing Dp(11)17/+ mice with heterozygous Rai1+/–_mice, we generated animals carrying a heterozygous duplication of 3 Mb containing Rai1, along with 18 other genes on one chromosome, and a null allele of Rai1 on the other chromosome [_Dp(11)17/Rai1– mice] (Figure 4A). Therefore the _Dp(11)17/Rai1–_mice, like the Dp(11)17/+ mice, retain 3 copies of each of those 18 other genes but differ in that they now have the normal disomic (n = 2) copy number of Rai1.
Mating scheme to normalize Rai1 and the correlation of Rai1 copy number changes and body weight. (A) Expected genotypes of the progeny from the mating of Dp(11)17/+ and Rai1+/– mice. Rai1 alleles are represented by a line with circles on it. The filled circles represent normal Rai1 gene, whereas the open circles represent the null copy of Rai1. Rai1 gene copy number is further indicated in brackets for each genotype. The 4 different genotypes were obtained at expected Mendelian ratios. Mice for each of the 4 genotypes were weighed every 2 weeks until 6 months of age. (B) Weight curves for female wild-type (open diamonds), Dp(11)17/+ (filled squares), Dp(11)17/Rai1–(open triangles), and Rai1+/–(filled circles) mice. (C) Data for male mice are shown. Each point represents the average of weights from at least 10 mice, and error bars are indicated. *P < 0.05.
Body weight normalization in Dp(11)17/Rai1– mice. Dp(11)17/+ mice are significantly underweight when compared with wild-type littermates (21). To explore the influence of Rai1 copy number on this particular phenotype, mice of the different genotypes were weighed biweekly from 1 to 6 months of age (Figure 4, B and C). Remarkably, Dp(11)17/Rai1– mice, carrying 2 copies of the Rai1 gene but 3 copies of the other 18 genes in the chromosome-engineered duplication interval, showed body weight similar to that of the wild-type animals, and their weight was significantly different from that of the Dp(11)17/+ and Rai1+/– mice. These data indicate that normalization of Rai1 gene copy number in the context of Dp(11)17 is sufficient to restore body weight to normal, in spite of all the other genes remaining trisomic in this genomic interval. This finding suggests that duplication of Rai1 is responsible for the reduced body weight phenotype of the Dp(11)17/+ mice. In addition, we have previously shown that homozygous Dp(11)17/Dp(11)17 animals (carrying 4 copies of the Rai1 gene), are even leaner than the heterozygous mice and display significant growth retardation (21). Furthermore, heterozygous Rai1+/– mice (23) are obese, as are mice in which this genomic interval was deleted to different extents, with the deletions always encompassing the Rai1 gene (21, 24). These data in aggregate indicate that Rai1 copy number and gene dosage are critical to body weight regulation.
Behavioral characterization as a function of Rai1 copy number variation. Dup(17)(p11.2p11.2) patients also have neurobehavioral abnormalities, including mild to borderline mental retardation, attention deficit disorder, hyperactivity, and autistic features (19). Despite the impossibility of recapitulating the complexity of human neurobehavioral phenotypes in mice, many of these phenotypes are associated with intermediate traits (also called endophenotypes) that can be analyzed in animal models (25). Such endophenotypes may result from chromosomal abnormalities or single gene defects or represent complex traits. In fact, Dp(11)17/+ mice exhibit elevated levels of anxiety, learning and memory deficits, and hyperactivity (22). Therefore, we next assessed the neurobehavioral phenotypic consequences of reconstituting Rai1 copy number to the normal disomic dosage in male mice.
Partial normalization of behaviors assayed by open-field testing. We subjected the Dp(11)17/_Rai1_– mice to the open-field test, used to assess exploratory activity and anxiety-related responses in a novel arena.
Dp(11)17/+ mice displayed less rearing behavior or vertical activity (Figure 5A) in the open field than wild-type mice.Although this difference in rearing was not quite statistically significant (P = 0.065), it is consistent with the statistically significant difference shown in Figure 2A. In contrast, Rai1+/– mice displayed more rearing than wild-type mice (P = 0.055), and Dp(11)17/Rai1– mice displayed rearing responses similar to those in wild-type mice (P = 0.459). Together these findings indicate that correcting Rai1 gene copy number normalized the rearing behavioral response of Dp(11)17/+ mice in the genetic backgrounds analyzed in the present study. Anxiety-related responses in the open field as measured by the center/total distance ratio (Figure 5B) clearly indicate that Dp(11)17/+ mice spend a lower proportion of their exploration in the center of the open field compared with wild-type mice (P < 0.05). The decreased center/total ratio for the Dp(11)17/+ mice replicates our previous findings of increased anxiety-related behaviors in Dp(11)17/+ animals (22) and is consistent with those data shown for the Dp(11)17/+ mice in Figure 2. Interestingly, the Dp(11)17/Rai1– mice displayed a similar center/total ratio compared with wild-type littermates, indicating that the increased anxiety-related behavior in the open field in the Dp(11)17/+ mice was attenuated by normalization of the Rai1 copy number. Rai1+/– mice also had a center/total ratio similar to that of wild-type littermates. Finally, replicating our previous reported results (22), and similar to the results presented in Figure 2, Dp(11)17/+ animals traveled significantly farther compared with wild-type littermates (P = 0.008) (Figure 5C). Consistent with the Dp(11)17/Df(11)17 mice, Dp(11)17/Rai1– mice were also more active than wild-type littermates (P < 0.05), indicating that this abnormality may not be totally related to copy number of Rai1. Although normalizing copy number of Rai1 did not significantly alter the response of Dp(11)17/+ mice, Rai1+/– mice were significantly less active (P < 0.05) compared with wild-type mice, suggesting that reduced Rai1 copy number does contribute to this behavioral response.
Correcting Rai1 gene dosage rescues behavioral deficits. Different parameters measured in the open field are represented for each of the 4 genotypes tested. (A) Vertical activity [F(3, 73) = 4.458; P = 0.06]. (B) Center/total distance ratio [F(3, 71) = 4.893; P < 0.004]. (C) Total distance (cm) [F(3, 73) = 9.498; P < 0.001]. Black bars, wild type; light gray bars, Dp(11)17/+; dark gray bars, Rai1+/–; white bars, Dp(11)17/Rai1–. Values represent mean ± SEM. Actual values are given in Supplemental Table 3. *P < 0.05, **P < 0.01, significant differences compared with wild-type littermates.
The light-dark exploration test, typically used to assess anxiety-related responses, and the prepulse inhibition test, used to assess sensorimotor gating, did not show any overall significant differences (P > 0.05) for any of the mice reported in this article (data not shown). A decrease in light-dark transitions and in startle response was previously reported for Dp(11)17/+ mice (22). As mice of different genetic backgrounds were used in the 2 studies, it is possible that there were modifiers present that affected the outcome of Rai1 copy number variation.
Learning and memory is normalized in Dp(11)17/Rai1– mice. We assessed learning and memory using a conditioned fear test based on the Pavlovian paradigm. As previously reported (22), Dp(11)17/+ mice displayed significantly less freezing during the context test than wild-type mice (P = 0.0005) (Figure 6A). However, the percentage of freezing observed for Dp(11)17/Rai1– mice was similar to that observed for wild-type mice (P > 0.05), clearly indicating that the contextual fear impairment is directly related to Rai1 copy number. Levels of freezing during the pre–conditioned stimulus (pre-CS) and CS phases were not significantly different among the various genotypes (P > 0.05) (Figure 6B).
Normalizing Rai1 gene dosage rescues learning and memory deficits. The percentages of freezing to the context [F(3, 73) = 6.573, P = 0.001] (A) and the sound cue (B) are represented for each of the 4 genotypes. Black bars: wild type; light gray bars: Dp(11)17/+; dark gray bars, Rai1+/–; white bars: Dp(11)17/Rai1–. Note that the abnormal freezing of Dp(11)17/+ animals is totally corrected in Dp(11)17/ Rai1– mice. The mean ± SEM values are presented. Actual values are given in Supplemental Table 3. **P < 0.001, significant difference compared with wild type.