Dissection of a Down syndrome-associated trisomy to separate the gene dosage-dependent and -independent effects of an extra chromosome

Abstract

As an aneuploidy, trisomy is associated with mammalian embryonic and postnatal abnormalities. Understanding the underlying mechanisms involved in mutant phenotypes is broadly important and may lead to new strategies to treat clinical manifestations in individuals with trisomies, such as trisomy 21 [Down syndrome (DS)]. Although increased gene dosage effects because of a trisomy may account for the mutant phenotypes, there is also the possibility that phenotypic consequences of a trisomy can arise because of the presence of a freely segregating extra chromosome with its own centromere, i.e. a 'free trisomy' independent of gene dosage effects. Presently, there are no reports of attempts to functionally separate these two types of effects in mammals. To fill this gap, here we describe a strategy that employed two new mouse models of DS, Ts65Dn;Df(17)2Yey/+ and Dp(16)1Yey/Df(16)8Yey. Both models carry triplications of the same 103 human chromosome 21 gene orthologs; however, only Ts65Dn;Df(17)2Yey/+ mice carry a free trisomy. Comparison of these models revealed the gene dosage-independent impacts of an extra chromosome at the phenotypic and molecular levels for the first time. They are reflected by impairments of Ts65Dn;Df(17)2Yey/+ males in T-maze tests when compared with Dp(16)1Yey/Df(16)8Yey males. Results from the transcriptomic analysis suggest the extra chromosome plays a major role in trisomy-associated expression alterations of disomic genes beyond gene dosage effects. This model system can now be used to deepen our mechanistic understanding of this common human aneuploidy and obtain new insights into the effects of free trisomies in other human diseases such as cancers.

Figure 1

Schematic illustration of Mmu16 and Mmu17 in four mouse mutants. As depicted, to separate the gene dosage-dependent and -independent effects of an extra chromosome, we generated Ts65Dn;Df(17)2/+ and Dp(16)1/Df(16)8 compound mouse mutants. Both mutants carry three copies of the Mir155-Zbtb21 region highlighted by black. Thus, there are no differences in genes or gene dosages between Ts65Dn;Df(17)2/+ and Dp(16)1/Df(16)8. However, only Ts65Dn;Df(17)2/+ has an extra chromosome (trisomy). 16, Mmu16; 17, Mmu17.

Figure 2

(A)–(D) Generation of Df(17)2/+ mice. (A) Strategy for generating Df(17)2. MICER vectors MHPN155l15 and MHPP338g19 were linearized with NdeI and HpaI, respectively, before targeting. E, EcoRI; H, HpaI. (B) Illustration of genomic locations of the BAC probes used in the FISH analysis. (C) FISH analysis of the metaphase spread prepared from the ES cells carrying Df(17)2. (D) Southern blot analysis of EcoRI-digested mouse tail DNA hybridized with probe 2B. Lane 1 and 2 represent Df(17)2/+ mice. (E)–(H) Generation of Df(16)8/+ mice. (E) Strategy for generating Df(16)8. MICER vectors MHPN121h17 and MHPP56i01 were linearized with SwaI and AflII, respectively, before targeting. N, NdeI; V, EcoRV. (F) Illustration of genomic locations of the BAC probes used in the FISH analysis. (G) FISH analysis of the metaphase spread prepared from the ES cells carrying Df(16)8/+. (H) Southern blot analysis of NdeI-digested mouse tail DNA hybridized with probe 2D. Lane 1 and 2 represent Df(16)8/+ mice. These results demonstrate that the desired deletions are present in the engineered mutants. 5′, 5′ HPRT fragment; 3′, 3′ HPRT fragment; N, neomycin-resistance gene; P, puromycin-resistance gene; Ty, Tyrosinase transgene; Ag, Agouti transgene; arrowhead, loxP site.

Figure 3

Agilent microarray CGH profile. DNA from (A) Ts65Dn;Df(17)2/+ and (B) Dp(16)1/Df(16)8 mice were used for the analysis. CGH profiles for Mmu16 and Mmu17 for each mouse are shown. The data represent the log2-transformed hybridization ratios of (A) Ts65Dn;Df(17)2/+ or (B) Dp(16)1/Df(16)8 mouse DNA versus WT mouse DNA.

Figure 4

(A) Nesting test. WT (males, n = 13; females, n = 17), Ts65Dn;Df(17)2/+ (males, n = 15; females, n = 18) and Dp(16)1/Df(16)8 (males, n = 15; females, n = 18) mice were supplied with a pre-weighed Nestlet in separate cages before the start of the dark cycle. The nesting results were recorded the next day. Each nest was assigned a score from 0 to 5 (0, nestlet untouched, no nest in the cage; 5, perfect nest, almost no nestlet remaining). Data are presented as the mean ± SEM for both sexes and for males and females separately. At the end of the test, the remaining Nestlet was weighed and recorded. Data are presented as the mean ± SEM for both sexes and for males and females separately. ^**, P < 0.01; ^*, P < 0.05. (B) T-maze test. WT (males, n = 16; females, n = 17), Ts65Dn;Df(17)2/+ (males, n = 15; females, n = 18) and Dp(16)1/Df(16)8 (males, n = 15; females, n = 18) mice were allowed to explore the T-maze for 10 min. Each entry into each arm was recorded. The alternation rate was calculated by dividing the number of correct alternations by the total possible alternations. The alternation rates are shown for both sexes, for males and females separately. Data are presented as the mean ± SEM. ^*, P < 0.05.

Figure 5

Analysis of differential expressions via RNA-seq-based transcriptome profiling of the cerebral cortex isolated from Ts65Dn;Df(17)2/+ (males, n = 5; females, n = 5), Dp(16)1/Df(16)8 (males, n = 5; females, n = 5) and WT control mice (males, n = 6; females, n = 6). (A) The heatmaps of the DE genes between Ts65Dn;Df(17)2/+ and WT as well as between Dp(16)1/Df(16)8 and WT. (B) The heatmaps of the DE genes between Ts65Dn;Df(17)2/+ and Dp(16)1/Df(16)8. (C) The Venn diagram illustrating the relationship between the higher-expressed and lower-expressed Ts65Dn;Df(17)2/+ genes versus the WT controls and the higher-expressed and lower-expressed Dp(16)1/Df(16)8 genes versus the WT controls (see Supplementary Material, Table S1 for the names of the DE genes represented in the heatmaps and the Venn diagram as well as Supplementary Material, Table S2 for the numbers of the DE genes in various categories).

Figure 6

(A) Open field test. WT (males, n = 10; females, n = 13), Ts65Dn (males, n = 7; females, n = 8) and Ts65Dn;Df(17)2/+ (males, n = 7; females, n = 10) mice were allowed to explore the open field arena for 10 min. Total path length (m) was analyzed in both sexes and in males and females separately. (B) T-maze test. WT (males, n = 10; females, n = 13), Ts65Dn (males, n = 7; females, n = 8) and Ts65Dn;Df(17)2/+ (males, n = 7; females, n = 10) mice were allowed to explore the T-maze for 10 min. Each entry into each arm was recorded. The alternation rate was calculated by dividing the number of correct alternations by the total possible alternations. The alternation rates are shown for both sexes and for males and females separately. (C) Morris water maze test. WT (males, n = 9; females, n = 12), Ts65Dn (males, n = 7; females, n = 8) and Ts65Dn;Df(17)2/+ (males, n = 7; females, n = 9) mice were compared using the Morris water maze test. During the hidden platform test from day 1 to 6, the path length of Ts65Dn, but not Ts65Dn;Df(17)2/+, was significantly longer than that of the WT mice when both sexes were calculated together (P < 0.001). In male mice, the path length of Ts65Dn, but not Ts65Dn;Df(17)2/+, was significantly longer than that of the WT mice (P < 0.01). In female mice, the path length of Ts65Dn was significantly longer than that of the WT mice (P < 0.01); meanwhile, the Ts65Dn;Df(17)2/+ mice showed a tendency to take a longer path but the post hoc analysis did not reveal significant difference for any trial days. Data are presented as the mean ± SEM. ^***, P < 0.001; ^**, P < 0.01; ^*, P < 0.05.