Deletion of the App-Runx1 region in mice models human partial monosomy 21

Abstract

Partial monosomy 21 (PM21) is a rare chromosomal abnormality that is characterized by the loss of a variable segment along human chromosome 21 (Hsa21). The clinical phenotypes of this loss are heterogeneous and range from mild alterations to lethal consequences, depending on the affected region of Hsa21. The most common features include intellectual disabilities, craniofacial dysmorphology, short stature, and muscular and cardiac defects. As a complement to human genetic approaches, our team has developed new monosomic mouse models that carry deletions on Hsa21 syntenic regions in order to identify the dosage-sensitive genes that are responsible for the symptoms. We focus here on the Ms5Yah mouse model, in which a 7.7-Mb region has been deleted from the App to Runx1 genes. Ms5Yah mice display high postnatal lethality, with a few surviving individuals showing growth retardation, motor coordination deficits, and spatial learning and memory impairments. Further studies confirmed a gene dosage effect in the Ms5Yah hippocampus, and pinpointed disruptions of pathways related to cell adhesion (involving App, Cntnap5b, Lgals3bp, Mag, Mcam, Npnt, Pcdhb2, Pcdhb3, Pcdhb4, Pcdhb6, Pcdhb7, Pcdhb8, Pcdhb16 and Vwf). Our PM21 mouse model is the first to display morphological abnormalities and behavioural phenotypes similar to those found in affected humans, and it therefore demonstrates the major contribution that the App-Runx1 region has in the pathophysiology of PM21.

Keywords: Aneuploidy; Learning and memory; Motor coordination; Mouse model.

Fig. 1.

Viability tests and anatomical characterization of foetuses at E18.5. (A-C) Pictures of neonates, 30 min after caesarean section. Healthy foetuses moved and turned pink in the first few minutes after delivery. (A) Wild-type and (B) cyanotic Ms5Yah littermates. (C) A Ms5Yah foetus that presented craniorachischisis. (D-G) Anatomical characterization. Lungs from a wild-type foetus at low (D) and high magnifications (F) presented opened alveoli as the foetus was breathing before euthanasia. Lungs from a Ms5Yah foetus that was unable to breathe at low (E) and high (G) magnifications presented collapsed alveoli. Scale bars: 3 mm (D,E); 400 µm (F,G).

Fig. 2.

Coordination capacities of newborn animals and development of their weight. (A) Righting reflex of wild-type (2n) and Ms5Yah newborn mice. Graphs plotting the time (s) to recover natural posture when animals were placed on their back. (B) Evolution of body weight (g) from 1 to 13 weeks of age. The legend is the same as that shown in A. (C) 10-week-old wild-type (bottom of the image) and Ms5Yah (top of the image) littermates. All graphs depict the mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001.

Fig. 3.

Locomotor coordination and muscular capacities. (A,B) Training phase of the rotarod test for wild-type (2n) and Ms5Yah mice. (A) Results are expressed as the time (s) that mice remained on an accelerating rod (4-40 rpm over 5 min) before falling. The legend given in C applies to panels A and B. (B) Mean rotational velocity (rpm) at the time of falling. (C) Test phase. The graphs plot the time (s) that mice stayed on the rod when tested at constant speeds between 4 and 40 rpm. (D) Notched bar test. The results are expressed as the percentage of hind paw errors made by mice when crossing the bar. (E) Four-paw grip test. These tests indicate that Ms5Yah show impaired motor coordination without alterations of muscular strength. All graphs depict the mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001.

Fig. 4.

Spatial learning and memory performances in the Morris water maze test. (A) Distance travelled to find the platform along acquisition (A1–A6) and reversal (Cued1 and Cued2) sessions for wild-type (2n) and Ms5Yah mice. (B) Corresponding swimming speeds. (C,D) Removal session. Mice were scored for the percentage of time spent in the different quadrants (NE, north east; SE, south east; SW, south west; NW, north west) of the Morris water maze (C) and the annulus crossing (D) for one single trial where the platform was removed. Ms5Yah mice showed delays in the acquisition of the task and travelled a longer distance to find the platform. In addition to spatial learning impairments, the removal session indicated a deficit in accurate spatial memory as Ms5Yah mice had difficulty in remembering the exact position of the platform. All graphs depict the mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001.

Fig. 5.

Microarray and quantitative RT-PCR expression analyses on hippocampi. (A) Clustering derived from statistically deregulated genes in Ms5Yah in comparison to expression in wild-type (2n) mice. Underexpression and overexpression are represented in green and red, respectively, and expression levels were calculated by comparison with the mean expression level of all arrays for each gene. (B) Expression profile of App-Runx1 genes referenced on chips. Most of the genes had a fold change ranging from 0.5 to 0.8. Some genes of the Krtap cluster presented a fold change above threshold, which was probably due to several Krtap RNAs hybridizing to the same probes. (C) Quantitative PCR expression study focusing on candidate genes for memory deficits of Ms5Yah animals. The results confirm the transcriptome analysis showing the underexpression of App, Olig1 and Olig2 genes of the App-Runx1 region, and the overexpression of Klk6. All graphs depict the mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001.