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. 2016 Jan 14:5:e11614.
doi: 10.7554/eLife.11614.

Genetic dissection of Down syndrome-associated congenital heart defects using a new mouse mapping panel

Eva Lana-Elola  1 , Sheona Watson-Scales  1 , Amy Slender  1 , Dorota Gibbins  1 , Alexandrine Martineau  1 , Charlotte Douglas  1 , Timothy Mohun  1 , Elizabeth Mc Fisher  2 , Victor Lj Tybulewicz  1   3
Affiliations

Genetic dissection of Down syndrome-associated congenital heart defects using a new mouse mapping panel

Eva Lana-Elola et al. Elife. .

Erratum in

Abstract

Down syndrome (DS), caused by trisomy of human chromosome 21 (Hsa21), is the most common cause of congenital heart defects (CHD), yet the genetic and mechanistic causes of these defects remain unknown. To identify dosage-sensitive genes that cause DS phenotypes, including CHD, we used chromosome engineering to generate a mapping panel of 7 mouse strains with partial trisomies of regions of mouse chromosome 16 orthologous to Hsa21. Using high-resolution episcopic microscopy and three-dimensional modeling we show that these strains accurately model DS CHD. Systematic analysis of the 7 strains identified a minimal critical region sufficient to cause CHD when present in 3 copies, and showed that it contained at least two dosage-sensitive loci. Furthermore, two of these new strains model a specific subtype of atrio-ventricular septal defects with exclusive ventricular shunting and demonstrate that, contrary to current hypotheses, these CHD are not due to failure in formation of the dorsal mesenchymal protrusion.

Keywords: Down syndrome; chromosome engineering; congenital heart defects; developmental biology; human biology; medicine; mouse; mouse genetics; stem cells.

Plain language summary

Down syndrome is a condition caused by having an extra copy of one of the 46 chromosomes found inside human cells. Specifically, instead of two copies, people with Down syndrome are born with three copies of chromosome 21. This results in many different effects, including learning and memory problems, heart defects and Alzheimer’s disease. Each of these different effects is caused by having a third copy of one or more of the approximately 230 genes found on chromosome 21. However, it is not known which of these genes cause any of these effects, and how an extra copy of the genes results in such changes. Now, Lana-Elola et al. have investigated which genes on chromosome 21 cause the heart defects seen in Down syndrome, and how those heart defects come about. This involved engineering a new strain of mouse that has an extra copy of 148 mouse genes that are very similar to 148 genes found on chromosome 21 in humans. Like people with Down syndrome, this mouse strain developed heart defects when it was an embryo. Using a series of six further mouse strains, Lana-Elola et al. then narrowed down the potential genes that, when in three copies, are needed to cause the heart defects, to a list of just 39 genes. Further experiments then showed that at least two genes within these 39 genes were required in three copies to cause the heart defects. The next step will be to identify the specific genes that actually cause the heart defects, and then work out how a third copy of these genes causes the developmental problems.

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Conflict of interest statement

No competing interests declared.

Figures

Figure 1.
Figure 1.. Generation of a mouse mapping panel of partial trisomies for DS phenotypes.
(a) Representation of Hsa21 and the regions of conserved synteny with orthologous regions on Mmu10, Mmu17 and Mmu16 (grey lines), indicating the coordinates of each mouse region. Diagram of Hsa21 shows the main cytogenetic bands (rectangles of different colors) and the centromere (oval). Black lines show the extent of the duplication in each of the mouse strains reported here, indicating the first and last genes at the ends of each duplication. The number of genes and extent in Megabases (Mb) of each duplicated region was calculated as described in Methods (b) CGH analysis of each duplication strain (2 mice analyzed/strain). Plots show log2-transformed hybridization ratios for DNA from each duplication strain versus C57BL/6JNimr. The duplicated regions would be expected to have a 1.5-fold increase in DNA content (log2 ratio = 0.58).
Figure 2.
Figure 2.. Dp1Tyb mice recapitulate the CHD seen in DS.
(a) Graph shows the percentage of CHD in Dp1Tyb embryonic hearts at E14.5 compared to wild-type (Wt) littermates; n = 26 Wt and 39 Dp1Tyb embryos. Significant difference to corresponding wild-type incidence (Fisher’s exact test) *p<0.05. (b) 3D HREM rendering of Wt and Dp1Tyb hearts, eroded from anterior to posterior to show the four-chamber view in 3 successive planes. Outflow tract defects such as double outlet right ventricle are observed in Dp1Tyb hearts (red arrow head indicates the aortic valve connecting to the right ventricle). (c) 3D reconstructions of Wt and Dp1Tyb E14.5 hearts with different types of CHD (red arrowheads): perimembranous ventricular septal defects (pVSD), muscular ventricular septal defect (mVSD), atrio-ventricular septal defect (AVSD). Ao, aorta; AoV, aortic valve; iAVC, inferior atrio-ventricular cushion; LV, left ventricle; MV, mitral valve; PT, pulmonary trunk; RV, right ventricle; sAVC, superior atrio-ventricular cushion; TV, tricuspid valve; VS, ventricular septum.
Figure 3.
Figure 3.. Genetic dissection of DS-associated CHD.
(a) Representation of Hsa21 showing region of conserved synteny with Mmu16 in grey and the extent of the duplications in Dp9Tyb, Dp2Tyb and Dp3Tyb in black. Graphs show the incidence of CHD in each strain; n = 22 Wt, 17 Dp9Tyb; n = 16 Wt, 26 Dp2Tyb and n = 26 Wt, 25 Dp3Tyb embryos. Significant difference to corresponding wild-type incidence (Fisher’s exact test) *P<0.05. ns, not significant. (b) 3D reconstructions of Wt and Dp3Tyb E14.5 hearts with different types of CHD (red arrowheads) as described in Figure 2c. (c) Representation of Hsa21 showing region of conserved synteny with Mmu16 in grey and the extent of the duplications in the indicated strains in black. Graphs shows incidence of CHD. n = 20 Wt, 21 Ts1Rhr; n = 15 Wt, 19 Dp4Tyb; n = 11 Wt, 20 Dp5Tyb and n = 28 Wt, 19 Dp6Tyb embryos analyzed. ns, not significant (Fisher’s exact test). iAVC, inferior atrio-ventricular cushion; LV, left ventricle; MV, mitral valve; RV, right ventricle; sAVC, superior atrio-ventricular cushion; TV, tricuspid valve; VS, ventricular septum.
Figure 4.
Figure 4.. Development of the DMP.
Left panels show a series of 3D four-chamber views of hearts at embryonic stages E11.5, 12.5, 13.5 and 14.5. Middle panels show a close up of the AV junction (frontal plane view corresponds to blue dashed box in the left panels) in the Isl1Cre/Rosa26RLacZ (Isl1Cre) and the Isl1nLacZ strains; β–galactosidase expression is pseudo-colored in red. Right panels show a sagittal view of the AV junction in E11.5 and E12.5 hearts and a transverse view in E13.5 and E14.5 hearts; sagittal and transverse planes shown as orange dashed line in left panels; β–galactosidase expression is pseudo-colored in red. In the middle and right panels the DMP is marked with a black dashed contour. The number of biological replicates used for each of the developmental stages, E11.5, E12.5, E13.5 and E14.5 respectively, were: n = 2, 2, 6 and 4 for the Isl1Cre/Rosa26RLacZ and n = 10, 9, 7 and 5 for the Isl1nLacZ. AoV, aortic valve; AVC, atrioventricular canal; iAVC, inferior atrioventricular cushion; MV, mitral valve; RA, right atrium; TV, tricuspid valve; VS, ventricular septum. Scale bar, 200 μm.
Figure 5.
Figure 5.. Largely normal development of the DMP in Dp1Tyb mice.
(a) 3D reconstruction of control Isl1nLacZ and Isl1nLacZ/Dp1Tyb E11.5 and E12.5 hearts analyzed using dual filter HREM. In the first two columns heart morphology is in grey and LacZ expression is pseudo-colored in red. Left panels show a transverse view of the developing heart and middle panels show a sagittal plane at E11.5 and a 4-chamber view at E12.5. The DMP is marked with a dashed black contour. Orientation is marked with 4 arrows (A, anterior; D, dorsal; L, left; P, posterior; R, right; V, ventral). Right image panels show two representative 3D reconstructions of the DMP for control and Dp1Tyb embryos at E11.5 and E12.5; the DMP is pseudo-colored in blue and surrounding dorsal mesenchyme is pseudo-colored in yellow. Graphs show the volume and shape factor of the DMP (n = 9 Wt and 9 Dp1Tyb E11.5 embryos, n = 9 Wt and 12 Dp1Tyb E12.5 embryos). Results are presented with measurements from each heart and as mean ± s.e.m. Statistical analysis with an unpaired Student’s t-test; ns, not significant, *P<0.05. (b) 3D HREM rendering of hearts from a single Wt and a single Dp1Tyb embryo at E14.5. The left images show the AV junction in short axis view from the ventricles towards the atria. Red arrowheads indicate the bridging leaflets of the common AV junction in the Dp1Tyb heart. The middle images show a 4-chamber view visualizing the ventricular septum. Red arrowhead indicates a pVSD in the Dp1Tyb heart. The right images show a more dorsal plane of the 4-chamber view to visualize the DMP, marked with a red dashed contour in both Wt and Dp1Tyb hearts. iAVC, inferior atrioventricular cushion; LV, left ventricle; MV, mitral valve; OFT, outflow tract; RA, right atrium; RV, right ventricle; SAN, sinoatrial node; sAVC, superior atrioventricular cushion; TV, tricuspid valve; VS, ventricular septum. Scale bar, 50 μm.
Figure 6.
Figure 6.. Genetic and mechanistic dissection of CHD in mouse models of DS.
(a) Representation of Hsa21 and the conserved region of synteny with the telomeric part of Mmu16 (grey line). Black lines indicate the extent of the duplications in Dp3Tyb and Ts1Rhr strains. Magnifications show the ends of the duplications indicating the 8 genes duplicated in Dp3Tyb but not Ts1Rhr mice. (b) Hsa21 and orthologous portion of Mmu16 (grey line), showing extent of duplication in Dp3Tyb (red line, this study) and Dp(16)4Yey (black line) (Liu et al., 2013). Also shown are the extent of partial trisomies of two individuals with DS who had a VSD (PM) or AVSD (BA) (blue lines) (Korbel et al., 2009). (c) Diagrams show a normal heart (right) and 3 types of AVSD depending on the relationship of the AV valves to the septal components and the presence/absence of the DMP (red). Defects in the DMP are seen in AVSD with atrial shunting (ostium primum) or in complete AVSD with both atrial and ventricular shunts. Dp1Tyb mice have AVSD with a ventricular shunt and the DMP is present (red boxes). AS, atrial septum; DMP, dorsal mesenchymal protrusion; LA, left atrium; LV, left ventricle; MV, mitral valve; RA, right atrium; RV, right ventricle; TV, tricuspid valve; VS, ventricular septum.
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