Craniofacial phenotypes in segmentally trisomic mouse models for Down syndrome

Trisomy for chromosome 21 (Chr 21) has profound effects on development that result in a constellation of phenotypes known as Down syndrome (DS). Distinctive craniofacial manifestations are among the few features common to all individuals with DS. The characteristic face of a person with DS results primarily from maldevelopment of the underlying craniofacial skeleton. The Ts65Dn mouse, which has segmental trisomy 16, producing dosage imbalance for about half the genes found on human Chr 21, exhibits specific skeletal malformations corresponding directly to the craniofacial dysmorphogenesis in DS. Here we demonstrate that Ts1Cje mice, which are at dosage imbalance for about 3/4 of the genes triplicated in Ts65Dn, demonstrate a very similar pattern of anomalies in the craniofacial skeleton. However, one characteristic of Ts65Dn mice, a broadening of the cranial vault contributing to brachycephaly, is not seen in Ts1Cje mice. These observations independently confirm that a dosage imbalance for mouse genes orthologous to those on human Chr 21 has corresponding effects in both species. The subtle differences in the craniofacial phenotypes of Ts1Cje and Ts65Dn mice have implications for elucidation of the mechanisms by which this aneuploidy disrupts development.     

Down syndrome mouse models Ts65Dn, Ts1Cje, and Ms1Cje/Ts65Dn exhibit variable severity of cerebellar phenotypes.

Two mouse models are widely used for Down syndrome (DS) research. The Ts65Dn mouse carries a small chromosome derived primarily from mouse chromosome 16, causing dosage imbalance for approximately half of human chromosome 21 orthologs. These mice have cerebellar pathology with direct parallels to DS. The Ts1Cje mouse, containing a translocated chromosome 16, is at dosage imbalance for 67% of the genes triplicated in Ts65Dn. We quantified cerebellar volume and granule cell and Purkinje cell density in Ts1Cje. Cerebellar volume was significantly affected to the same degree in Ts1Cje and Ts65Dn, despite that Ts1Cje has fewer triplicated genes. However, dosage imbalance in Ts1Cje had little effect on granule cell and Purkinje cell density. Several mice with dosage imbalance for the segment of the Ts65Dn chromosome not triplicated in Ts1Cje had phenotypes that contrasted with those in Ts1Cje. These observations do not readily differentiate between two prevalent hypotheses for gene action in DS.     

Discovery and genetic localization of Down syndrome cerebellar phenotypes using the Ts65Dn mouse

Down syndrome (DS) is the most common genetic cause of mental retardation and affects many aspects of brain development. DS individuals exhibit an overall reduction in brain size with a disproportionately greater reduction in cerebellar volume. The Ts65Dn mouse is segmentally trisomic for the distal 12-15 Mb of mouse chromosome 16, a region that shows perfect conserved linkage with human chromosome 21, and therefore provides a genetic model for DS. In this study, high resolution magnetic resonance imaging and histological analysis demonstrate precise neuro- anatomical parallels between the DS and the Ts65Dn cerebellum. Cerebellar volume is significantly reduced in Ts65Dn mice due to reduction of both the internal granule layer and the molecular layer of the cerebellum. Granule cell number is further reduced by a decrease in cell density in the internal granule layer. Despite these changes in Ts65Dn cerebellar structure, motor deficits have not been detected in several tests. Reduction in granule cell density in Ts65Dn mice correctly predicts an analogous pathology in humans; a significant reduction in granule cell density in the DS cerebellum is reported here for the first time. The candidate region of genes on chromosome 21 affecting cerebellar development in DS is therefore delimited to the subset of genes whose orthologs are at dosage imbalance in Ts65Dn mice, providing the first localization of genes affecting a neuroanatomical phenotype in DS. The application of this model for analysis of developmental perturbations is extended by the accurate prediction of DS cerebellar phenotypes.     

Differential effects of trisomy on brain shape and volume in related aneuploid mouse models

Down syndrome (DS) results from inheritance of three copies of human chromosome 21 (Hsa21). Individuals with DS have a significantly smaller brain size overall and a disproportionately small cerebellum. The small cerebellum is seen in Ts65Dn mice, which have segmental trisomy for orthologs of about half the genes on Hsa21 and provide a genetic model for DS. While small cerebellar size is well-established in mouse and humans, much less is known about the shape of the brain in trisomy. Here we conduct a morphometric analysis of the whole brain and cerebellum in Ts65Dn mice and show that the differences with euploid littermates are largely a function of volume and not of shape. This is not the case in two aneuploid mouse models that have fewer genes orthologous to Hsa21 than Ts65Dn. Ts1Rhr is trisomic for genes corresponding to the so-called Down syndrome critical region (DSCR), which was purported to contain a dosage sensitive gene or genes responsible for many phenotypes of DS. Ms1Rhr is monosomic for the same segment. These models show effects on cerebellum and overall brain that are different from each other and from Ts65Dn. These models can help to identify the contributions of genes from different regions of the chromosome on this and other aspects of brain development in trisomy.     

Characterization of the cardiac phenotype in neonatal Ts65Dn mice.

The Ts65Dn mouse is the most-studied of murine models for Down syndrome. Homology between the triplicated murine genes and those on human chromosome 21 correlates with shared anomalies of Ts65Dn mice and Down syndrome patients, including congenital heart defects. Lethality is associated with inheritance of the T65Dn chromosome, and anomalies such as right aortic arch with Kommerell's diverticulum and interrupted aortic arch were found in trisomic neonates. The incidence of gross vascular abnormalities was 17% in the trisomic population. Histological analyses revealed interventricular septal defects and broad foramen ovale, while immunohistochemistry showed abnormal muscle composition in the cardiac valves of trisomic neonates. These findings confirm that the gene imbalance present in Ts65Dn disrupts crucial pathways during cardiac development. The candidate genes for congenital heart defects that are among the 104 triplicated genes in Ts65Dn mice are, therefore, implicated in the dysregulation of normal cardiogenic pathways in this model.     

Parallels of craniofacial maldevelopment in Down syndrome and Ts65Dn mice.

Mouse genetic models can be used to dissect molecular mechanisms that result in human disease. This approach requires detection and demonstration of compelling parallels between phenotypes in mouse and human. Ts65Dn mice are at dosage imbalance for many of the same genes duplicated in trisomy 21 or Down syndrome (DS), the most common live-born human aneuploidy. Analysis of the craniofacial skeleton of Ts65Dn mice using three-dimensional morphometric methods demonstrates an absolute correspondence between Ts65Dn and DS craniofacial dysmorphology, a distinctive and completely penetrant DS phenotype. The genes at dosage imbalance in Ts65Dn are localized to a small region of mouse chromosome 16 and, by comparative mapping, to the corresponding region of human Chromosome 21, providing independent experimental data supporting the contribution of genes in this region to this characteristic DS phenotype. This analysis establishes precise parallels in human and mouse skull phenotypes resulting from dosage imbalance for the same genes, revealing strong conservation of the evolved developmental genetic program that underlies mammalian skull morphology and validating the use of this mouse model in the analysis of this important DS phenotype. This evolutionary conservation further establishes the mouse as a valid model for a wide range of syndromes producing craniofacial maldevelopment.     

Trisomy for the Down syndrome 'critical region' is necessary but not sufficient for brain phenotypes of trisomic mice

Trisomic Ts65Dn mice show direct parallels with many phenotypes of Down syndrome (DS), including effects on the structure of cerebellum and hippocampus. A small segment of Hsa21 known as the 'DS critical region' (DSCR) has been held to contain a gene or genes sufficient to cause impairment in learning and memory tasks involving the hippocampus. To test this hypothesis, we developed Ts1Rhr and Ms1Rhr mouse models that are, respectively, trisomic and monosomic for this region. Here, we show that trisomy for the DSCR alone is not sufficient to produce the structural and functional features of hippocampal impairment that are seen in the Ts65Dn mouse and DS. However, when the critical region is returned to normal dosage in trisomic Ms1Rhr/Ts65Dn mice, performance in the Morris water maze is identical to euploid, demonstrating that this region is necessary for the phenotype. Thus, although the prediction of the critical region hypothesis was disproved, novel gene dosage effects were identified, which help to define how trisomy for this segment of the chromosome contributes to phenotypes of DS.     

Down syndrome gene dosage imbalance on cerebellum development

Down syndrome (DS) is a chromosomal disorder whereby genes on chromosome 21 are present in three copies. This gene copy imbalance is thought to be responsible for a number of debilitating conditions experienced by individuals with DS. Amongst these is a reduced cerebellar volume, or cerebellar hypoplasia, which is believed to contribute to the perturbation of fine motor control. Mouse models of DS (such as Ts65Dn, Ts1Cje, Tc1) exhibit a cerebellar phenotype similar to that of individuals with DS and which primarily manifests as a disruption of the density of the granule cell layer. Dissecting which of the three-copy genes are responsible for this phenotype (the primary gene dosage effect) has been a task undertaken by researchers working with various segmental trisomies and transgenic mice. It is generally agreed that, when expressed, three-copy genes of trisomic mice are expressed at around 1.5 times that of the same genes in euploid (wild-type) mice. However, amongst these studies there does not appear to be a consensus on the nature and extent of differential expression of two-copy genes in trisomic mice-the secondary dosage effect. Much of this variation may have to do with the stage of development investigated and the nature and complexity of the tissue (i.e. whole brain versus the cerebellum). The recent discovery that trisomic granule cell precursors are less sensitive to sonic hedgehog-induced proliferation has opened up another avenue for the identification of three-copy genes responsible for the cerebellar phenotype. It is hoped that further investigation of this phenomenon, together with new mouse segmental trisomies and transgenics, will reveal the cause of the proliferation deficit and allow for potential treatment.     

Cardiovascular Development and Survival During Gestation in the Ts65Dn Mouse Model for Down Syndrome

The Ts65Dn mouse model for Down syndrome (DS) exhibits many phenotypes seen in human DS. Previous research has revealed a reduced rate of transmission of the T65Dn marker chromosome in neonates. To analyze potential fetal loss, litters from trisomic females at 10.5dpc through 14.5dpc were genotyped. No significant differences from the expected Mendelian ratio were found in transmission of T65Dn at any stage. Cardiovascular defects found in trisomic neonates are associated with formation of pharyngeal arch arteries. Vessel tracing was used to identify anomalies in 10.5dpc, 11.5dpc, and 13.5dpc embryos. Comparison of trisomic versus euploid embryos injected with India ink revealed delay and abnormality in cardiovascular development in trisomic embryos at each stage. Through the analysis of transmission rate and cardiovascular development in embryonic mice, we learn more about prenatal mortality and the origins of cardiac abnormality in the Ts65Dn mice to assist in understanding cardiovascular malformation associated with DS. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Upregulation of β-catenin expression in down syndrome model Ts65Dn mouse brain

Trisomy of human chromosome 21 (Hsa 21) causes the pathological characteristics of Down syndrome (DS). Little is known about the mechanisms by which trisomy 21 affects the expression of genes on other chromosomes. Using a mouse model of DS, the Ts65Dn mouse, we have performed mRNA and protein measurements to identify genes on chromosomes not syntenic with Hsa 21 whose expression is affected by the presence of three copies of genes between loci Mrpl39 and Znf295 on mouse chromosome 16 (Mmu 16). We report the upregulation of β-catenin, located on mouse chromosome 9 (Mmu 9) in Ts65Dn brain. Using immunocytochemistry on Ts65Dn and control mouse brain tissue, we observed a striking increase in β-catenin expression specifically in the endothelial cells lining the cerebral blood vessels of the Ts65Dn mice. Since β-catenin is involved in cell–cell adhesion, upregulation of this protein in DS may alter adherens protein interactions that are involved in the normal functions of endothelial cells. Elevated β-catenin might be responsible for altered endothelial cell function/s leading to the impairment of brachial flow velocity observed in DS.     

Embryonic and not maternal trisomy causes developmental attenuation in the Ts65Dn mouse model for Down syndrome

Trisomy 21 results in Down syndrome (DS) and causes phenotypes that may result from alterations of developmental processes. The Ts65Dn mouse is the most widely used genetic and phenotypic model for DS. We used over 1,500 offspring from Ts65Dn and two nontrisomic genetically similar control strains to investigate the influence of trisomy on developmental alterations and number of offspring. For the first time, we demonstrate gross developmental attenuation of Ts65Dn trisomic offspring at embryonic day (E) 9.5 and E13.5 and show that the major determinant of the developmental changes is segmental trisomy of the embryo and not the trisomic maternal uterine environment. Maternal alleles of nontrisomic genes linked to Pde6b may also influence the development of Ts65Dn offspring. Both developmental attenuation and the contribution of trisomic and nontrisomic genes are important components in the genesis of DS phenotypes. Developmental Dynamics 239:1645–1653, 2010. © 2010 Wiley‐Liss, Inc.     

Behavioral assessment of the Ts65Dn mouse,a model for down syndrome: Altered behavior in the elevated plus maze and open field

The Ts65Dn mouse carries a partial trisomy for mouse chromosome 16 in a region that has high homology to the Down syndrome (DS) region of human chromosome 21 and is, thus, a potential animal model of DS. The focus of the present study was to begin to characterize the behavioral phenotype of this mouse to assess its usefulness as a model of aspects of the DS phenotype. The behavior of Ts65Dn and littermate control mice was assessed in the elevated plus maze, lighted and dark open field, and a step-down passive avoidance task. The behavior of Ts65Dn mice in these tests differed considerably from the nontrisomic controls. In the elevated plus maze, Ts65Dn had more total arm visits than controls, showed a higher percentage of arm visits to the open arms than control mice, and showed no preference for the closed arms. Ts65Dn mice were more active in both open-field situations, regardless of light condition, and ventured into the center of the arena more than controls. Lighting in the open field had moderate effects on the activity of the Ts65Dn mice, but control mice were, as expected, much more active in the dark than the light. The trisomic mice learned and retained the step-down passive avoidance task in the same number of trials as the controls. Overall, these data indicate that Ts65Dn mice are more active than control mice in two testing situations. Most striking is the finding that the Ts65Dn mice were much less responsive to variations in environmental cues to which normal mice are quite sensitive. These data not only begin to characterize systematically the Ts65Dn phenotype, but also raise several interesting issues about the sources of the aberrant behaviors observed in these mice.     

The effects of piracetam on cognitive performance in a mouse model of Down's syndrome

Piracetam is a nootropic agent that has been shown to improve cognitive performance in a number of animal model systems. Piracetam is reported to be used widely as a means of improving cognitive function in children with Down's syndrome (DS). In order to provide a preclinical assessment of the potential efficacy of piracetam, we examined the effects of a dose range of piracetam in the Ts65Dn mouse model of DS. Ts65Dn mice are trisomic for a region of mouse chromosome 16 with homology to human chromosome 21. Daily piracetam treatment at doses of 0, 75, 150, and 300 mg/kg ip was initiated in 6-week-old male Ts65Dn and euploid control mice. Following 4 weeks of treatment, mice were tested in the visible and hidden-platform components of the Morris water maze and were placed overnight in computerized activity chambers to assess effects on overall activity. Piracetam treatment was continued through the 4 weeks of testing. In control mice, 75 and 150 mg/kg/day piracetam improved performance in both the visible- and hidden-platform tasks. Although low doses of piracetam reduced search time in the visible-platform component in Ts65Dn mice, all piracetam doses prevented trial-related improvements in performance in Ts65Dn mice. The 300-mg/kg/day-piracetam dose was associated with a reversal of the nocturnal spontaneous hyperactivity in Ts65Dn. These data do not provide support for piracetam treatment for individuals with DS.     

Mice containing a human chromosome 21 model behavioral impairment and cardiac anomalies of Down's syndrome

Trisomy 21 (Ts21) is the most common live-born human aneuploidy; it results in a constellation of features known as Down's syndrome (DS). Ts21 is the most frequent cause of congenital heart defects and the leading genetic cause of mental retardation. To investigate the gene dosage effects of an extra copy of human chromosome 21 (Chr 21) on various phenotypes, we used microcell-mediated chromosome transfer to create embryonic stem (ES) cells containing Chr 21. ES cell lines retaining Chr 21 as an independent chromosome were used to produce chimeric mice with a substantial contribution from Chr 21-containing cells. Fluorescence in situ hybridization and PCR-based DNA analysis revealed that Chr 21 was substationally intact but had sustained a small deletion. The freely segregating Chr 21 was lost during development in some tissues, resulting in a panel of chimeric mice with various mosaicism as regards retention of the Chr 21. These chimeric mice showed a high correlation between retention of Chr 21 in the brain and impairment in learning or emotional behavior by open-field, contextual fear conditioning and forced swim tests. Hypoplastic thymus and cardiac defects, i.e. double outlet right ventricle and riding aorta, were observed in a considerable number of chimeric mouse fetuses with a high contribution of Chr 21. These chimeric mice mimic a wide variety of phenotypic traits of DS, revealing the utility of mice containing Chr 21 as unique models for DS and for the identification of genes responsible for DS.     

Duplication of the entire 22.9 Mb human chromosome 21 syntenic region on mouse chromosome 16 causes cardiovascular and gastrointestinal abnormalities

Down syndrome is caused by a genomic imbalance of human chromosome 21 which is mainly observed as trisomy 21. The regions on human chromosome 21 are syntenically conserved in three regions on mouse chromosomes 10, 16 and 17. Ts65Dn mice, the most widely used model for Down syndrome, are trisomic for approximately 56.5% of the human chromosome 21 syntenic region on mouse chromosome 16. To generate a more complete trisomic mouse model of Down syndrome, we have established a 22.9 Mb duplication spanning the entire human chromosome 21 syntenic region on mouse chromosome 16 in mice using Cre/loxP-mediated long-range chromosome engineering. The presence of the intact duplication in mice was confirmed by fluorescent in situ hybridization and BAC-based array comparative genomic hybridization. The expression levels of the genes within the duplication interval reflect gene-dosage effects in the mutant mice. The cardiovascular and gastrointestinal phenotypes of the mouse model were similar to those of patients with Down syndrome. This new mouse model represents a powerful tool to further understand the molecular and cellular mechanisms of Down syndrome.     

Dosage-dependent over-expression of genes in the trisomic region of Ts1Cje mouse model for Down syndrome

Down syndrome (DS) is the most common chromosomally caused form of mental retardation and is caused by trisomy of chromosome 21. The over-expression of genes located on the trisomic region has been assumed to be responsible for the phenotypic abnormalities of DS, but this hypothesis has not been confirmed fully and the very existence of gene dosage effects has been called into question. We have therefore investigated global gene expression profiles in Ts1Cje, a mouse model for DS that displays learning deficits and has a segmental trisomy of chromosome 16 orthologous to a segment of human chromosome 21 spanning from Sod1 to Znf295. DNA microarray analyses of six Ts1Cje and six normal littermate (2N) mouse brains at postnatal day 0 with probe sets representing approximately 11,300 genes revealed that the number of expressed genes and their identities in Ts1Cje mice were almost same in 2N mice. Notably, the expression levels of most genes in the trisomic region were increased approximately 1.5-fold, and the top 24 most consistently over-expressed genes in the Ts1Cje mice were all located in the trisomic region. In contrast, the expression levels of genes on other chromosomes or the euploid region of chromosome 16 were largely the same (1.0-fold) in Ts1Cje and 2N mice. These results indicate that the genes in the trisomic region of Ts1Cje are over-expressed in a dosage-dependent manner and are implicated in the molecular pathogenesis of DS.     

Abnormal expression of synaptic proteins and neurotrophin-3 in the Down syndrome mouse model Ts65Dn

Down syndrome (DS) results from triplication of the whole or distal part of human chromosome 21. Persons with DS suffer from deficits in learning and memory and cognitive functions in general, and, starting from early development, their brains show dendritic and spine structural alterations and cell loss. These defects concern many cortical brain regions as well as the hippocampus, which is known to play a critical role in memory and cognition. Most of these abnormalities are reproduced in the mouse model Ts65Dn, which is partially trisomic for the mouse chromosome 16 that is homologous to a portion of human chromosome 21. Thus, Ts65Dn is widely utilized as an animal model of DS. To better understand the molecular defects underlying the cognitive and particularly the memory impairments of DS, we investigated whether the expression of several molecules known to play critical roles in long-term synaptic plasticity and long-term memory in a variety of species is dysregulated in either the neonatal brain or adult hippocampus of Ts65Dn mice. We found abnormal expression of the synaptic proteins synaptophysin, microtubule-associated protein 2 (MAP2) and cyclin-dependent kinase 5 (CDK5) and of the neurotrophin-3 (NT-3). Both the neonatal brain and adult hippocampus revealed significant abnormalities. These results suggest that a dysregulation in the expression of neurotrophins as well as proteins involved in synaptic development and plasticity may play a potential role in the neural pathology of DS in humans.     

Systemic pathology in aged mouse models of Down's syndrome and Alzheimer's disease

Down's syndrome (DS) in humans is caused by trisomy of chromosome 21 (HSA 21). DS patients have a variety of pathologies, including mental retardation and an unusually high incidence of leukemia or lymphoma such as megakaryocytic leukemia. Individuals with DS develop the characteristic neuropathological hallmarks of Alzheimer's disease (AD) in early adulthood, generally by the fourth decade of life. There are several mouse models of DS that have a segmental trisomy of mouse chromosome 16 (MMU 16) with triplicated genes orthologous to HSA 21. These mice display neurodegeneration similar to DS. Although brain pathology in DS models is known, little information is available about other organs. We studied the extraneural pathology in aged DS mice (Ts65Dn, Ts2 and Ts1Cje aged 8 to 24 months) as well as other mouse models of neurodegeneration, including presenilin (PS), amyloid-β precursor protein (APP), and tau (hTau and JNPL) transgenic mice. An increased incidence of peripheral amyloidosis, positive for amyloid A (AA) but not amyloid-β peptide (Aβ), was found in APP over-expressing and tauopathic mice as compared to non-transgenic (ntg) littermates or to DS mouse models. A higher incidence of lymphoma was found in the DS models, including Ts1Cje that is trisomic for a small segment of MMU 16 not including the App gene, but not in the APP over-expressing mice, suggesting that high APP expression is not the cause of lymphoma in DS. The occurrence of lymphomas in mouse DS models is of interest in relation to the increased incidence of malignant conditions in human DS.