Handling Editor: Monica J. Justice
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- Fig. 1.
Graphical depiction of the Ts65Dn timeline. Ts65Dn was generated in 1990 as the first viable postnatal model of DS. In 2007, a complete embryonic characterization was published (Chakrabarti et al., 2007) describing multiple neurodevelopmental deficits. In 2010, it was reported that Ts65Dn had been corrected to contain only the wild-type Pde6b preventing the recessive retinal degeneration present in the original animals. Since 2010, there have been two continuously breeding strains of Ts65Dn at The Jackson Laboratory: 1924, which is the originally developed line with retinal degeneration; and 5252, a line genetically identical but without the recessive allele. The cohorts of animals analyzed in this paper are designated with their strain number and year the animals analyzed were imported from The Jackson Laboratory. The 52522014 line was used to analyze the DMs and MWM reported in Olmos-Serrano et al. (2016a,b).
- Fig. 2.
Gross brain size and cortical thickness during embryonic development. (A,B) Measurements of M-L (A) and R-C lengths (B) of the developing telencephalon in 19242017 mice at E14.5 (n=7 and 8, respectively) and E15.5 (n=6 and 6). (C) 19242017 pallial thickness measurements at E14.5 and E15.5. (D) M-L width measurements in the 5252 cohorts at E14.5 (52522014, n=4 euploids and 5 trisomic; 52522019, n=7 euploid and 9 trisomic; and 5252Cryo2010, n=10 euploid and 10 trisomic). (E) R-C length in 5252 cohorts at E14.5. (F) Pallial thickness measurements in different 5252 cohorts at E14.5. (G) M-L width in the 5252 cohorts at E15.5 (52522014, n=3 euploid and 3 trisomic; 52522015, n=6 euploid and 5 trisomic; 52522019, n=21 euploid and 6 trisomic; 5252Cryo2010, n=9 euploid and 6 trisomic). (H) R-C length at E15.5 in the 5252 cohorts. (I) Pallial thickness measurements in the 5252 cohorts at E15.5. (J) Representative images of euploid and trisomic cortical walls in developing embryos at E13.5, E14.5 and E15.5 in the 5252Cryo2010 cohort. (K,L) At E14.5, when trisomic animals showed significant differences in total pallial thickness, both 52522019 and 5252Cryo2010 show significant changes in the IZ but no significant differences in the VZ/SVZ or CP. All comparisons were made using an unpaired two-tailed Student's t-test with a probability level of P<0.05 (*) considered statistically significant. Box plots are mean±Q1 and Q3.
- Fig. 3.
Neurodevelopmental deficits across embryonic time in 5252Cryo2010. (A) Gross measurements of 5252Cryo2010 mice at E13.5 (n=10 euploid and 9 trisomic), E14.5 (n=10 euploid and 10 trisomic) and E15.5 (n=9 euploid and 6 trisomic) compared to their euploid littermates (dashed line). (B) Thickness of the VZ/SVZ, IZ, CP and total pallium of the 5252Cryo2010 trisomic embryo as a percentage of their euploid littermates (dashed line) at E13.5, E14.5 and E15.5. The CP has not yet developed at E13.5. (C,D) Representative images of the developing cortex stained with PH3 to mark mitotic cells and Tbr2 to mark intermediate precursors. (E) Number of abventricular PH3+ cells in euploid and trisomic embryos at E13.5, E14.5 and E15.5. (F) Number of Tbr2+ intermediate precursor cells in euploid and trisomic embryos at E13.5, E14.5 and E15.5. All comparisons were made using an unpaired two-tailed Student's t-test with a probability level of P<0.05 (*) considered statistically significant and were conducted between trisomic embryos and their euploid controls at each time point. Box plots are median±Q1 and Q3.
- Fig. 4.
Gross body and brain measurements across the lifespan in different colonies of Ts65Dn. (A) Body weight of euploids and Ts65Dn at P15 (5252Cryo2010, n=7 and 9, respectively; 52522019, n=19 and 5, respectively), P30 (5252Cryo2010, n=8 and 7, respectively; 52522019, n=10 and 10, respectively), and P60 (5252Cryo2010, n=8 and 8, respectively; 52522019 n=5 and 5, respectively). (B,C) R-C (B) and M-L (C) length measured as shown in Fig. 2E at P15 (5252Cryo2010, n=7 euploid and 9 trisomic; 52522019 n=19 euploid and 5 trisomic), P30 (5252Cryo2010, n=8 and 7; 52522019, n=10 and 10), and P60 (5252Cryo2010, n=6 and 6; 52522019, n=5 and 5). (D) Hindlimb reflex measured at P15 (5252Cryo2010, n=20 euploid and 15; trisomic 52522019, n=19 euploid and 5 trisomic), P30 (5252Cryo2010, n=8 euploid and 7 trisomic; 52522019, n=10 euploid and 10 trisomic) and P60 (5252Cryo2010, n=12 euploid and 12 trisomic; 52522019, n=5 euploid and 5 trisomic). # indicates that all animals scored a value of zero for that group. Data are mean±s.e.m. (E) Representative image of euploid (left) and 5252Cryo2010 (right) brains at P30, with the location of where measurements were taken indicated. Boxplots are median±Q1 and Q3, with individual data points. All comparisons were made using an unpaired two-way Student's t-test between trisomic and relative euploid controls, with a probability level of P<0.05 (*) considered statistically significant.
- Fig. 5.
Different iterations of the Ts65Dn colony show varied changes in oligodendrocyte maturation. (A) Percentage of mature oligodendrocytes (CC1+/Olig2+ cells) within the corpus callosum of 5252Cryo2010 males at P15 (n=5 euploid and 4 trisomic), P21 (n=5 euploid and 5 trisomic), P30 (n=4 euploid and 5 trisomic) and P60 (n=6 euploid and 6 trisomic). (B) Percentage of CC1+/Olig2+ cells counted within the corpus callosum of euploid and trisomic 52522015 male mice at P15 (n=7 and 4, respectively), P21 (n=9 and 7, respectively), and P60 (n=3 and 3, respectively), with no significant differences between genotypes. (C) Percentage of CC1+/Olig2+ mature oligodendrocytes in the corpus callosum of 52522019 euploid and trisomic males at P15 (n=5 and 5, respectively), P30 (n=5 and 5, respectively) and P60 (n=5 and 5, respectively). (D,E) Representative images of the corpus callosum stained with CC1, Ng2 and Olig2 in both euploid (D) and 5252Cryo2010 (E) animals. (F,G) RT-qPCR gene expression analysis for myelin-related genes in the corpus callosum (F) and cortex (G) of 5252Cryo2010 (n=5) and 52522019 (n=5) mice at P30, with trisomic expression levels normalized to the expression of the respective gene in euploid controls (n=5 and 5). Data are mean±s.e.m. Boxplots are median±Q1 and Q3, with individual data points. All comparisons were made using an unpaired two-way Student's t-test between trisomic and relative euploid controls, with a probability level of P<0.05 (*) considered statistically significant.
- Fig. 6.
Hippocampal and cerebellar cell densities assessed in 5252Cryo2010, 52522015, and 52522019 males at P60. (A) Density of NeuN+ cells in the CA1 and CA3 regions of the hippocampus of 5252Cryo2010 (n=5 euploid and 5 trisomic), 52522015 (n=5 euploid and 3 trisomic) and 52522019 (n=5 euploid and 5 trisomic) cohorts normalized to their euploid controls as a percent of control. (B) NeuN+ cell density in lobule III and lobule IV/V of the cerebellum of 5252Cryo2010 (n=5 euploid and 5 trisomic), 52522015 (n=5 euploid and 3 trisomic) and 52522019 (n=5 euploid and 5 trisomic). (C-F) Representative images of NeuN staining in CA1 (C), CA3 (D), lobule III (E) and lobule IV/V (F). Boxplots are median±Q1 and Q3, with individual data points and trisomic values normalized to respective euploid controls. All comparisons were made using an unpaired two-way Student's t-test between trisomic and relative euploid controls. No comparison yielded P<0.05.
- Fig. 7.
DMs measured at the age animals acquired a particular skill. (A-J) Boxplots of DM tasks for euploid males (n=20), trisomic males (n=15), euploid females (n=20) and trisomic females (n=12) from the 5252Cryo2010 cohort. (K) Table illustrating behaviors that were significantly delayed in 52522014 Ts65Dn mice in previous publications (Olmos-Serrano et al., 2016b; Aziz et al., 2018) versus the presently studied 5252Cryo2010 mice. Arrows indicate behaviors that trisomic animals were significantly delayed in. Boxplots are median±Q1 and Q3, with individual data points. All comparisons were made using an unpaired two-way Student's t-test between trisomic animals and same-sex euploid controls with a probability level of P<0.05 (*) considered statistically significant.
- Fig. 8.
5252Cryo2010 mice show impaired spatial learning on the MWM task compared to euploid controls. (A) The latency to reach the cued or hidden platform across consecutive days of the task was significantly impacted by genotype (n=24 euploid and 24 trisomic, F1, 46=11.28, P=0.002). (B) Latency to reach platform was not influenced by sex for trisomic mice (n=12 euploid and 12 trisomic, F1,22=1.11, P>0.999). (C) Probe trial conducted after the hidden acquisition phase shows time spent in the target quadrant, with 5252Cryo2010 mice spending significantly less time in the target quadrant than controls (P=0.004). (D,E) Swimming speed and distance traveled across days during the hidden acquisition phase. There was no effect of genotype on velocity (F1, 46=0.006, P=0.936) but trisomic mice swam greater total distances compared to euploids (F1, 46=9.557, P=0.003). (F,G) Scatter plots of total time spent in target quadrant on probe trial versus cumulative DM score for males (F) and females (G). Pearson correlation coefficient calculated for each sex shows no relationship between the acquisition of DMs and performance on the MWM test. Data are mean±s.e.m. for each genotype. Repeated measured two-way ANOVAs and post-hoc Sidak multiple comparisons tests were used to compare trisomic performance to controls. A probability level of P<0.05 (*) was considered statistically significant for all measures.
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