Mohammed Selloum

A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains

BackgroundThe mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.ResultsWe undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems.ConclusionsComparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.

High-throughput discovery of novel developmental phenotypes.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.

Mouse functional genomics requires standardization of mouse handling and housing conditions

The study of mouse models is crucial for the functional annotation of the human genome. The recent improvements in mouse genetics now moved the bottleneck in mouse functional genomics from the generation of mutant mice lines to the phenotypic analysis of these mice lines. Simple, validated, and reproducible phenotyping tests are a prerequisite to improving this phenotyping bottleneck. We analyzed here the impact of simple variations in animal handling and housing procedures, such as cage density, diet, gender, length of fasting, as well as site (retro-orbital vs. tail), timing, and anesthesia used during venipuncture, on biochemical, hematological, and metabolic/endocrine parameters in adult C57BL/6J mice. Our results, which show that minor changes in procedures can profoundly affect biological variables, underscore the importance of establishing uniform and validated animal procedures to improve reproducibility of mouse phenotypic data.

Genetic background determines metabolic phenotypes in the mouse

To evaluate the contribution of genetic background to phenotypic variation, we compared a large range of biochemical and metabolic parameters at different ages of four inbred mice strains, C57BL/6J, 129SvPas, C3HeB/FeJ, and Balb/cByJ. Our results demonstrate that important metabolic, hematologic, and biochemical differences exist between these different inbred strains. Most of these differences are gender independent and are maintained or accentuated throughout life. It is therefore imperative that the genetic background is carefully defined in phenotypic studies. Our results also argue that certain backgrounds are more suited to study a given physiologic phenomenon, as distinct mouse strains have a different propensity to develop particular biochemical, hematologic, and metabolic abnormalities. These genetic differences can furthermore be exploited to identify new genes/proteins that contribute to phenotypic abnormalities. The choice of the genetic background in which to generate and analyze genetically engineered mutant mice is important as it is, together with environmental factors, one of the most important contributors to the variability of phenotypic results.

Altered lipoprotein metabolism in P2Y13 knockout mice

The purinergic receptor P2Y(13) has been shown to play a role in the uptake of holo-HDL particles in in vitro hepatocyte experiments. In order to determine the role of P2Y(13) in lipoprotein metabolism in vivo, we ablated the expression of this gene in mice. Here we show that P2Y(13) knockout mice have lower fecal concentrations of neutral sterols (-27%±2.1% in males) as well as small decreases in plasma HDL (-13.1%±3.2% in males; -17.5%±4.0% in females) levels. In addition, significant decreases were detected in serum levels of fatty acids and glycerol in female P2Y(13) knockout mice. Hepatic mRNA profiling analyses showed increased expression of SREBP-regulated cholesterol and fatty acid biosynthesis genes, while fatty acid β-oxidation genes were significantly decreased. Liver gene signatures also identified changes in PPARα-regulated transcript levels. With the exception of a small increase in bone area, P2Y(13) knockout mice do not show any additional major abnormalities, and display normal body weight, fat mass and lean body mass. No changes in insulin sensitivity and oral glucose tolerance could be detected. Taken together, our experiments assess a role for the purinergic receptor P2Y(13) in the regulation of lipoprotein metabolism and demonstrate that modulating its activity could be of benefit to the treatment of dyslipidemia in people.

Conditional depletion of intellectual disability and Parkinsonism candidate gene ATP6AP2 in fly and mouse induces cognitive impairment and neurodegeneration

ATP6AP2, an essential accessory component of the vacuolar H+ ATPase (V-ATPase), has been associated with intellectual disability (ID) and Parkinsonism. ATP6AP2 has been implicated in several signalling pathways; however, little is known regarding its role in the nervous system. To decipher its function in behaviour and cognition, we generated and characterized conditional knockdowns of ATP6AP2 in the nervous system of Drosophila and mouse models. In Drosophila, ATP6AP2 knockdown induced defective phototaxis and vacuolated photoreceptor neurons and pigment cells when depleted in eyes and altered short- and long-term memory when depleted in the mushroom body. In mouse, conditional Atp6ap2 deletion in glutamatergic neurons (Atp6ap2Camk2aCre/0 mice) caused increased spontaneous locomotor activity and altered fear memory. Both Drosophila ATP6AP2 knockdown and Atp6ap2Camk2aCre/0 mice presented with presynaptic transmission defects, and with an abnormal number and morphology of synapses. In addition, Atp6ap2Camk2aCre/0 mice showed autophagy defects that led to axonal and neuronal degeneration in the cortex and hippocampus. Surprisingly, axon myelination was affected in our mutant mice, and axonal transport alterations were observed in Drosophila. In accordance with the identified phenotypes across species, genome-wide transcriptome profiling of Atp6ap2Camk2aCre/0 mouse hippocampi revealed dysregulation of genes involved in myelination, action potential, membrane-bound vesicles and motor behaviour. In summary, ATP6AP2 disruption in mouse and fly leads to cognitive impairment and neurodegeneration, mimicking aspects of the neuropathology associated with ATP6AP2 mutations in humans. Our results identify ATP6AP2 as an essential gene for the nervous system.

Ptchd1 deficiency induces excitatory synaptic and cognitive dysfunctions in mouse

Synapse development and neuronal activity represent fundamental processes for the establishment of cognitive function. Structural organization as well as signalling pathways from receptor stimulation to gene expression regulation are mediated by synaptic activity and misregulated in neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID). Deleterious mutations in the PTCHD1 (Patched domain containing 1) gene have been described in male patients with X-linked ID and/or ASD. The structure of PTCHD1 protein is similar to the Patched (PTCH1) receptor; however, the cellular mechanisms and pathways associated with PTCHD1 in the developing brain are poorly determined. Here we show that PTCHD1 displays a C-terminal PDZ-binding motif that binds to the postsynaptic proteins PSD95 and SAP102. We also report that PTCHD1 is unable to rescue the canonical sonic hedgehog (SHH) pathway in cells depleted of PTCH1, suggesting that both proteins are involved in distinct cellular signalling pathways. We find that Ptchd1 deficiency in male mice (Ptchd1−/y) induces global changes in synaptic gene expression, affects the expression of the immediate-early expression genes Egr1 and Npas4 and finally impairs excitatory synaptic structure and neuronal excitatory activity in the hippocampus, leading to cognitive dysfunction, motor disabilities and hyperactivity. Thus our results support that PTCHD1 deficiency induces a neurodevelopmental disorder causing excitatory synaptic dysfunction.

Mouse models of 17q21.31 microdeletion and microduplication syndromes highlight the importance of Kansl1 for cognition

Koolen-de Vries syndrome (KdVS) is a multi-system disorder characterized by intellectual disability, friendly behavior, and congenital malformations. The syndrome is caused either by microdeletions in the 17q21.31 chromosomal region or by variants in the KANSL1 gene. The reciprocal 17q21.31 microduplication syndrome is associated with psychomotor delay, and reduced social interaction. To investigate the pathophysiology of 17q21.31 microdeletion and microduplication syndromes, we generated three mouse models: 1) the deletion (Del/+); or 2) the reciprocal duplication (Dup/+) of the 17q21.31 syntenic region; and 3) a heterozygous Kansl1 (Kans1+/-) model. We found altered weight, general activity, social behaviors, object recognition, and fear conditioning memory associated with craniofacial and brain structural changes observed in both Del/+ and Dup/+ animals. By investigating hippocampus function, we showed synaptic transmission defects in Del/+ and Dup/+ mice. Mutant mice with a heterozygous loss-of-function mutation in Kansl1 displayed similar behavioral and anatomical phenotypes compared to Del/+ mice with the exception of sociability phenotypes. Genes controlling chromatin organization, synaptic transmission and neurogenesis were upregulated in the hippocampus of Del/+ and Kansl1+/- animals. Our results demonstrate the implication of KANSL1 in the manifestation of KdVS phenotypes and extend substantially our knowledge about biological processes affected by these mutations. Clear differences in social behavior and gene expression profiles between Del/+ and Kansl1+/- mice suggested potential roles of other genes affected by the 17q21.31 deletion. Together, these novel mouse models provide new genetic tools valuable for the development of therapeutic approaches.

The homeodomain factor Gbx1 is required for locomotion and cell specification in the dorsal spinal cord

Dorsal horn neurons in the spinal cord integrate and relay sensory information to higher brain centers. These neurons are organized in specific laminae and different transcription factors are involved in their specification. The murine homeodomain Gbx1 protein is expressed in the mantle zone of the spinal cord at E12.5-13.5, correlating with the appearance of a discernable dorsal horn around E14 and eventually defining a narrow layer in the dorsal horn around perinatal stages. At postnatal stages, Gbx1 identifies a specific subpopulation of GABAergic neurons in the dorsal spinal cord. We have generated a loss of function mutation for Gbx1 and analyzed its consequences during spinal cord development. Gbx1−/− mice are viable and can reproduce as homozygous null mutants. However, the adult mutant mice display an altered gait during forward movement that specifically affects the hindlimbs. This abnormal gait was evaluated by a series of behavioral tests, indicating that locomotion is impaired, but not muscle strength or motor coordination. Molecular analysis showed that the development of the dorsal horn is not profoundly affected in Gbx1−/− mutant mice. However, analysis of terminal neuronal differentiation revealed that the proportion of GABAergic inhibitory interneurons in the superficial dorsal horn is diminished. Our study unveiled a role for Gbx1 in specifying a subset of GABAergic neurons in the dorsal horn of the spinal cord involved in the control of posterior limb movement.

Increased H3K9 methylation and impaired expression of Protocadherins are associated with the cognitive dysfunctions of the Kleefstra syndrome

Abstract Kleefstra syndrome, a disease with intellectual disability, autism spectrum disorders and other developmental defects is caused in humans by haploinsufficiency of EHMT1. Although EHMT1 and its paralog EHMT2 were shown to be histone methyltransferases responsible for deposition of the di-methylated H3K9 (H3K9me2), the exact nature of epigenetic dysfunctions in Kleefstra syndrome remains unknown. Here, we found that the epigenome of Ehmt1+/− adult mouse brain displays a marked increase of H3K9me2/3 which correlates with impaired expression of protocadherins, master regulators of neuronal diversity. Increased H3K9me3 was present already at birth, indicating that aberrant methylation patterns are established during embryogenesis. Interestingly, we found that Ehmt2+/− mice do not present neither the marked increase of H3K9me2/3 nor the cognitive deficits found in Ehmt1+/− mice, indicating an evolutionary diversification of functions. Our finding of increased H3K9me3 in Ehmt1+/− mice is the first one supporting the notion that EHMT1 can quench the deposition of tri-methylation by other Histone methyltransferases, ultimately leading to impaired neurocognitive functioning. Our insights into the epigenetic pathophysiology of Kleefstra syndrome may offer guidance for future developments of therapeutic strategies for this disease.