Publications by authors named "Marie-Christine Birling"

41 Publications

Droplet digital PCR or quantitative PCR for in-depth genomic and functional validation of genetically altered rodents.

Methods 2021 Apr 7. Epub 2021 Apr 7.

PHENOMIN-Institut Clinique de la Souris, CELPHEDIA, CNRS, INSERM, Université de Strasbourg, Illkirch-Graffenstaden, Strasbourg, 67404, France. Electronic address:

Gene targeting and additive (random) transgenesis have proven to be powerful technologies with which to decipher the mammalian genome. With the advent of CRISPR/Cas9 genome editing, the ability to inactivate or modify the function of a gene has become even more accessible. However, the impact of each generated modification may be different from what was initially desired. Minimal validation of mutant alleles from genetically altered (GA) rodents remains essential to guarantee the interpretation of experimental results. The protocol described here combines design strategies for genomic and functional validation of genetically modified alleles with droplet digital PCR (ddPCR) or quantitative PCR (qPCR) for target DNA or mRNA quantification. In-depth analysis of the results obtained with GA models through the analysis of target DNA and mRNA quantification is also provided, to evaluate which pitfalls can be detected using these two methods, and we propose recommendations for the characterization of different type of mutant allele (knock-out, knock-in, conditional knock-out, FLEx, IKMC model or transgenic). Our results also highlight the possibility that mRNA expression of any mutated allele can be different from what might be expected in theory or according to common assumptions. For example, mRNA analyses on knock-out lines showed that nonsense-mediated mRNA decay is generally not achieved with a critical-exon approach. Likewise, comparison of multiple conditional lines crossed with the same CreER deleter showed that the inactivation outcome was very different for each conditional model. DNA quantification by ddPCR of G0 to G2 generations of transgenic rodents generated by pronuclear injection showed an unexpected variability, demonstrating that G1 generation rodents cannot be considered as established lines.
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http://dx.doi.org/10.1016/j.ymeth.2021.04.001DOI Listing
April 2021

Targeting the RHOA pathway improves learning and memory in adult Kctd13 and 16p11.2 deletion mouse models.

Mol Autism 2021 01 13;12(1). Epub 2021 Jan 13.

Université de Strasbourg, CNRS, INSERM, Institut de Génétique Biologie Moléculaire et Cellulaire - UMR 7104 - U1258, IGBMC, 1 rue Laurent Fries, 67404, Illkirch Cedex, France.

Background: Gene copy number variants play an important role in the occurrence of neurodevelopmental disorders. Particularly, the deletion of the 16p11.2 locus is associated with autism spectrum disorder, intellectual disability, and several other features. Earlier studies highlighted the implication of Kctd13 genetic imbalance in 16p11.2 deletion through the regulation of the RHOA pathway.

Methods: Here, we generated a new mouse model with a small deletion of two key exons in Kctd13. Then, we targeted the RHOA pathway to rescue the cognitive phenotypes of the Kctd13 and 16p11.2 deletion mouse models in a pure genetic background. We used a chronic administration of fasudil (HA1077), an inhibitor of the Rho-associated protein kinase, for six weeks in mouse models carrying a heterozygous inactivation of Kctd13, or the deletion of the entire 16p11.2 BP4-BP5 homologous region.

Results: We found that the small Kctd13 heterozygous deletion induced a cognitive phenotype similar to the whole deletion of the 16p11.2 homologous region, in the Del/+ mice. We then showed that chronic fasudil treatment can restore object recognition memory in adult heterozygous mutant mice for Kctd13 and for 16p11.2 deletion. In addition, learning and memory improvement occurred in parallel to change in the RHOA pathway.

Limitations: The Kcdt13 mutant line does not recapitulate all the phenotypes found in the 16p11.2 Del/+ model. In particular, the locomotor activity was not altered at 12 and 18 weeks of age and the object location memory was not defective in 18-week old mutants. Similarly, the increase in locomotor activity was not modified by the treatment in the 16p11.2 Del/+ mouse model, suggesting that other loci were involved in such defects. Rescue was observed only after four weeks of treatment but no long-term experiment has been carried out so far. Finally, we did not check the social behaviour, which requires working in another hybrid genetic background.

Conclusion: These findings confirm KCTD13 as one target gene causing cognitive deficits in 16p11.2 deletion patients, and the relevance of the RHOA pathway as a therapeutic path for 16p11.2 deletion. In addition, they reinforce the contribution of other gene(s) involved in cognitive defects found in the 16p11.2 models in older mice.
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http://dx.doi.org/10.1186/s13229-020-00405-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805198PMC
January 2021

Evidence that corticofugal propagation of ALS pathology is not mediated by prion-like mechanism.

Prog Neurobiol 2021 May 9;200:101972. Epub 2020 Dec 9.

Inserm UMR_S 1118, Centre de Recherche en Biomédecine de Strasbourg, Faculté de Médecine, Université de Strasbourg, 67 000, Strasbourg, France. Electronic address:

Amyotrophic lateral sclerosis (ALS) arises from the combined degeneration of motor neurons (MN) and corticospinal neurons (CSN). Recent clinical and pathological studies suggest that ALS might start in the motor cortex and spread along the corticofugal axonal projections (including the CSN), either via altered cortical excitability and activity or via prion-like propagation of misfolded proteins. Using mouse genetics, we recently provided the first experimental arguments in favour of the corticofugal hypothesis, but the mechanism of propagation remained an open question. To gain insight into this matter, we tested here the possibility that the toxicity of the corticofugal projection neurons (CFuPN) to their targets could be mediated by their cell autonomous-expression of an ALS causing transgene and possible diffusion of toxic misfolded proteins to their spinal targets. We generated a Crym-CreER mouse line to ablate the SOD1 transgene selectively in CFuPN. This was sufficient to fully rescue the CSN and to limit spasticity, but had no effect on the burden of misfolded SOD1 protein in the spinal cord, MN survival, disease onset and progression. The data thus indicate that in ALS corticofugal propagation is likely not mediated by prion-like mechanisms, but could possibly rather rely on cortical hyperexcitability.
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http://dx.doi.org/10.1016/j.pneurobio.2020.101972DOI Listing
May 2021

Reliable and robust droplet digital PCR (ddPCR) and RT-ddPCR protocols for mouse studies.

Methods 2020 Jul 25. Epub 2020 Jul 25.

PHENOMIN-Institut Clinique de la Souris, CELPHEDIA, CNRS, INSERM, Université de Strasbourg, Illkirch-Graffenstaden, Strasbourg 67404, France. Electronic address:

Droplet digital PCR (ddPCR) is a recent method developed for the quantification of nucleic acids sequences. It is an evolution of PCR methodology incorporating two principal differences: a PCR reaction is performed in thousands of water-oil emulsion droplets and fluorescence is measured at the end of PCR amplification. It leads to the precise and reproducible quantification of DNA and RNA sequences. Here, we present quantitative methods for DNA and RNA analysis using Bio-Rad QX100 or QX200 systems, respectively. The aim of these methods is to provide useful molecular tools for validating genetically altered animal models such as those subject to CRISPR/Cas9 genome editing, as well for expression or CNV studies. A standard procedure for simultaneous DNA and RNA extraction adapted for mouse organs is also described. These methods were initially designed for mouse studies but also work for samples from other species like rat or human. In our lab, thousands of samples and hundreds of target genes from genetically altered lines were examined using these methods. This large dataset was analyzed to evaluate technical optimizations and limitations. Finally, we propose additional recommendations to be included in dMIQE (Minimum information for publication of quantitative digital PCR experiments) guidelines when using ddPCR instruments.
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http://dx.doi.org/10.1016/j.ymeth.2020.07.004DOI Listing
July 2020

Universal Southern blot protocol with cold or radioactive probes for the validation of alleles obtained by homologous recombination.

Methods 2020 Jun 26. Epub 2020 Jun 26.

The Mary Lyon Centre, MRC Harwell Institute, Harwell Campus, Didcot, Oxon OX11 0RD, UK. Electronic address:

The widespread availability of recombineered vectors and gene targeted embryonic stem cells from large-scale repositories facilitates the generation of mouse models for functional genetic studies. Southern blotting validates the structure of these targeted alleles produced by homologous recombination, as well as indicating any additional integrations of the vector into the genome. Traditionally this technique employs radioactively-labelled probes; however, there are many laboratories that are restricted in their use of radioactivity. Here, we present a widely applicable protocol for Southern blot analysis using cold probes and alternative procedures employing radioactive probes. Furthermore, the probes are designed to recognise standardised regions of gene-targeting cassettes and so represent universally applicable reagents for assessing allelic integrity.
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http://dx.doi.org/10.1016/j.ymeth.2020.06.011DOI Listing
June 2020

Targeting Morphine-Responsive Neurons: Generation of a Knock-In Mouse Line Expressing Cre Recombinase from the Mu-Opioid Receptor Gene Locus.

eNeuro 2020 May/Jun;7(3). Epub 2020 May 29.

Department of Psychiatry, McGill University, Douglas Hospital Research Centre, Montreal, Quebec H4H 1R3, Canada

The mu-opioid receptor (MOR) modulates nociceptive pathways and reward processing, and mediates the strong analgesic and addictive properties of both medicinal as well as abused opioid drugs. MOR function has been extensively studied, and tools to manipulate or visualize the receptor protein are available. However, circuit mechanisms underlying MOR-mediated effects are less known, because genetic access to MOR-expressing neurons is lacking. Here we report the generation of a knock-in -Cre mouse line, which allows targeting and manipulating MOR opioid-responsive neurons. A cDNA encoding a T2A cleavable peptide and Cre recombinase fused to enhanced green fluorescent protein (EGFP/Cre) was inserted downstream of the gene sequence. The resulting Cre line shows intact gene transcription. MOR and EGFP/Cre proteins are coexpressed in the same neurons, and localized in cytoplasmic and nuclear compartments, respectively. MOR signaling is unaltered, demonstrated by maintained DAMGO-induced G-protein activation, and MOR function is preserved as indicated by normal morphine-induced analgesia, hyperlocomotion, and sensitization. The Cre recombinase efficiently drives the expression of Cre-dependent reporter genes, shown by local virally mediated expression in the medial habenula and brain-wide fluorescence on breeding with tdTomato reporter mice, the latter showing a distribution patterns typical of MOR expression. Finally, we demonstrate that optogenetic activation of MOR neurons in the ventral tegmental area of -Cre mice evokes strong avoidance behavior, as anticipated from the literature. The -Cre line is therefore an excellent tool for both mapping and functional studies of MOR-positive neurons, and will be of broad interest for opioid, pain, and addiction research.
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http://dx.doi.org/10.1523/ENEURO.0433-19.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266138PMC
May 2020

Modeling Down syndrome in animals from the early stage to the 4.0 models and next.

Prog Brain Res 2020 22;251:91-143. Epub 2019 Oct 22.

Université de Strasbourg, CNRS, INSERM, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France; Université de Strasbourg, CNRS, INSERM, PHENOMIN Institut Clinique de la Souris, Illkirch, France. Electronic address:

The genotype-phenotype relationship and the physiopathology of Down Syndrome (DS) have been explored in the last 20 years with more and more relevant mouse models. From the early age of transgenesis to the new CRISPR/CAS9-derived chromosomal engineering and the transchromosomic technologies, mouse models have been key to identify homologous genes or entire regions homologous to the human chromosome 21 that are necessary or sufficient to induce DS features, to investigate the complexity of the genetic interactions that are involved in DS and to explore therapeutic strategies. In this review we report the new developments made, how genomic data and new genetic tools have deeply changed our way of making models, extended our panel of animal models, and increased our understanding of the neurobiology of the disease. But even if we have made an incredible progress which promises to make DS a curable condition, we are facing new research challenges to nurture our knowledge of DS pathophysiology as a neurodevelopmental disorder with many comorbidities during ageing.
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http://dx.doi.org/10.1016/bs.pbr.2019.08.001DOI Listing
December 2020

Conditional switching of KIF2A mutation provides new insights into cortical malformation pathogeny.

Hum Mol Genet 2020 03;29(5):766-784

Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67400 Illkirch, France.

By using the Cre-mediated genetic switch technology, we were able to successfully generate a conditional knock-in mouse, bearing the KIF2A p.His321Asp missense point variant, identified in a subject with malformations of cortical development. These mice present with neuroanatomical anomalies and microcephaly associated with behavioral deficiencies and susceptibility to epilepsy, correlating with the described human phenotype. Using the flexibility of this model, we investigated RosaCre-, NestinCre- and NexCre-driven expression of the mutation to dissect the pathophysiological mechanisms underlying neurodevelopmental cortical abnormalities. We show that the expression of the p.His321Asp pathogenic variant increases apoptosis and causes abnormal multipolar to bipolar transition in newborn neurons, providing therefore insights to better understand cortical organization and brain growth defects that characterize KIF2A-related human disorders. We further demonstrate that the observed cellular phenotypes are likely to be linked to deficiency in the microtubule depolymerizing function of KIF2A.
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http://dx.doi.org/10.1093/hmg/ddz316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104682PMC
March 2020

Transgenic mouse models expressing human and macaque prion protein exhibit similar prion susceptibility on a strain-dependent manner.

Sci Rep 2019 10 30;9(1):15699. Epub 2019 Oct 30.

Centro de Investigación en Sanidad Animal (INIA-CISA), 28130, Valdeolmos, Madrid, Spain.

Cynomolgus macaque has been used for the evaluation of the zoonotic potential of prion diseases, especially for classical-Bovine Spongiform Encephalopathy (classical-BSE) infectious agent. PrP amino acid sequence is considered to play a key role in the susceptibility to prion strains and only one amino acid change may alter this susceptibility. Macaque and human-PrP sequences have only nine amino acid differences, but the effect of these amino acid changes in the susceptibility to dissimilar prion strains is unknown. In this work, the transmissibility of a panel of different prions from several species was compared in transgenic mice expressing either macaque-PrP (TgMac) or human-PrP (Hu-Tg340). Similarities in the transmissibility of most prion strains were observed suggesting that macaque is an adequate model for the evaluation of human susceptibility to most of the prion strains tested. Interestingly, TgMac were more susceptible to classical-BSE strain infection than Hu-Tg340. This differential susceptibility to classical-BSE transmission should be taken into account for the interpretation of the results obtained in macaques. It could notably explain why the macaque model turned out to be so efficient (worst case model) until now to model human situation towards classical-BSE despite the limited number of animals inoculated in the laboratory experiments.
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http://dx.doi.org/10.1038/s41598-019-52155-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821920PMC
October 2019

Severe head dysgenesis resulting from imbalance between anterior and posterior ontogenetic programs.

Cell Death Dis 2019 10 24;10(11):812. Epub 2019 Oct 24.

Université de Strasbourg, Inserm, IRFAC/UMR-S1113, FMTS, 67200, Strasbourg, France.

Head dysgenesis is a major cause of fetal demise and craniofacial malformation. Although mutations in genes of the head ontogenetic program have been reported, many cases remain unexplained. Head dysgenesis has also been related to trisomy or amplification of the chromosomal region overlapping the CDX2 homeobox gene, a master element of the trunk ontogenetic program. Hence, we investigated the repercussion on head morphogenesis of the imbalance between the head and trunk ontogenetic programs, by means of ectopic rostral expression of CDX2 at gastrulation. This caused severe malformations affecting the forebrain and optic structures, and also the frontonasal process associated with defects in neural crest cells colonization. These malformations are the result of the downregulation of genes of the head program together with the abnormal induction of trunk program genes. Together, these data indicate that the imbalance between the anterior and posterior ontogenetic programs in embryos is a new possible cause of head dysgenesis during human development, linked to defects in setting up anterior neuroectodermal structures.
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http://dx.doi.org/10.1038/s41419-019-2040-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813351PMC
October 2019

The neuroanatomy of Eml1 knockout mice, a model of subcortical heterotopia.

J Anat 2019 09 7;235(3):637-650. Epub 2019 Jun 7.

INSERM UMR S-1270, Paris, France.

The cerebral cortex is a highly organized structure responsible for advanced cognitive functions. Its development relies on a series of steps including neural progenitor cell proliferation, neuronal migration, axonal outgrowth and brain wiring. Disruption of these steps leads to cortical malformations, often associated with intellectual disability and epilepsy. We have generated a new resource to shed further light on subcortical heterotopia, a malformation characterized by abnormal neuronal position. We describe here the generation and characterization of a knockout (KO) mouse model for Eml1, a microtubule-associated protein showing mutations in human ribbon-like subcortical heterotopia. As previously reported for a spontaneous mouse mutant showing a mutation in Eml1, we observe severe cortical heterotopia in the KO. We also observe abnormal progenitor cells in early corticogenesis, likely to be the origin of the defects. EML1 KO mice on the C57BL/6N genetic background also appear to present a wider phenotype than the original mouse mutant, showing additional brain anomalies, such as corpus callosum abnormalities. We compare the anatomy of male and female mice and also study heterozygote animals. This new resource will help unravel roles for Eml1 in brain development and tissue architecture, as well as the mechanisms leading to severe subcortical heterotopia.
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http://dx.doi.org/10.1111/joa.13013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704242PMC
September 2019

TUBG1 missense variants underlying cortical malformations disrupt neuronal locomotion and microtubule dynamics but not neurogenesis.

Nat Commun 2019 05 13;10(1):2129. Epub 2019 May 13.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67400, Illkirch, France.

De novo heterozygous missense variants in the γ-tubulin gene TUBG1 have been linked to human malformations of cortical development associated with intellectual disability and epilepsy. Here, we investigated through in-utero electroporation and in-vivo studies, how four of these variants affect cortical development. We show that TUBG1 mutants affect neuronal positioning, disrupting the locomotion of new-born neurons but without affecting progenitors' proliferation. We further demonstrate that pathogenic TUBG1 variants are linked to reduced microtubule dynamics but without major structural nor functional centrosome defects in subject-derived fibroblasts. Additionally, we developed a knock-in Tubg1 mouse model and assessed consequences of the mutation. Although centrosomal positioning in bipolar neurons is correct, they fail to initiate locomotion. Furthermore, Tubg1 animals show neuroanatomical and behavioral defects and increased epileptic cortical activity. We show that Tubg1 mice partially mimic the human phenotype and therefore represent a relevant model for further investigations of the physiopathology of cortical malformations.
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http://dx.doi.org/10.1038/s41467-019-10081-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6513894PMC
May 2019

Cbs overdosage is necessary and sufficient to induce cognitive phenotypes in mouse models of Down syndrome and interacts genetically with Dyrk1a.

Hum Mol Genet 2019 05;28(9):1561-1577

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.

Identifying dosage-sensitive genes is a key to understand the mechanisms underlying intellectual disability in Down syndrome (DS). The Dp(17Abcg1-Cbs)1Yah DS mouse model (Dp1Yah) shows cognitive phenotypes that need to be investigated to identify the main genetic driver. Here, we report that three copies of the cystathionine-beta-synthase gene (Cbs) in the Dp1Yah mice are necessary to observe a deficit in the novel object recognition (NOR) paradigm. Moreover, the overexpression of Cbs alone is sufficient to induce deficits in the NOR test. Accordingly, overexpressing human CBS specifically in Camk2a-expressing neurons leads to impaired objects discrimination. Altogether, this shows that Cbs overdosage is involved in DS learning and memory phenotypes. To go further, we identified compounds that interfere with the phenotypical consequence of CBS overdosage in yeast. Pharmacological intervention in Tg(CBS) mice with one selected compound restored memory in the NOR test. In addition, using a genetic approach, we demonstrated an epistatic interaction between Cbs and Dyrk1a, another human chromosome 21-located gene (which encodes the dual-specificity tyrosine phosphorylation-regulated kinase 1a) and an already known target for DS therapeutic intervention. Further analysis using proteomic approaches highlighted several molecular pathways, including synaptic transmission, cell projection morphogenesis and actin cytoskeleton, that are affected by DYRK1A and CBS overexpression. Overall, we demonstrated that CBS overdosage underpins the DS-related recognition memory deficit and that both CBS and DYRK1A interact to control accurate memory processes in DS. In addition, our study establishes CBS as an intervention point for treating intellectual deficiencies linked to DS.
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http://dx.doi.org/10.1093/hmg/ddy447DOI Listing
May 2019

A new mouse model of ARX dup24 recapitulates the patients' behavioral and fine motor alterations.

Hum Mol Genet 2018 06;27(12):2138-2153

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg, 67404 Illkirch, France.

The aristaless-related homeobox (ARX) transcription factor is involved in the development of GABAergic and cholinergic neurons in the forebrain. ARX mutations have been associated with a wide spectrum of neurodevelopmental disorders in humans, among which the most frequent, a 24 bp duplication in the polyalanine tract 2 (c.428_451dup24), gives rise to intellectual disability, fine motor defects with or without epilepsy. To understand the functional consequences of this mutation, we generated a partially humanized mouse model carrying the c.428_451dup24 duplication (Arxdup24/0) that we characterized at the behavior, neurological and molecular level. Arxdup24/0 males presented with hyperactivity, enhanced stereotypies and altered contextual fear memory. In addition, Arxdup24/0 males had fine motor defects with alteration of reaching and grasping abilities. Transcriptome analysis of Arxdup24/0 forebrains at E15.5 showed a down-regulation of genes specific to interneurons and an up-regulation of genes normally not expressed in this cell type, suggesting abnormal interneuron development. Accordingly, interneuron migration was altered in the cortex and striatum between E15.5 and P0 with consequences in adults, illustrated by the defect in the inhibitory/excitatory balance in Arxdup24/0 basolateral amygdala. Altogether, we showed that the c.428_451dup24 mutation disrupts Arx function with a direct consequence on interneuron development, leading to hyperactivity and defects in precise motor movement control and associative memory. Interestingly, we highlighted striking similarities between the mouse phenotype and a cohort of 33 male patients with ARX c.428_451dup24, suggesting that this new mutant mouse line is a good model for understanding the pathophysiology and evaluation of treatment.
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http://dx.doi.org/10.1093/hmg/ddy122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985730PMC
June 2018

Nox4 genetic inhibition in experimental hypertension and metabolic syndrome.

Arch Cardiovasc Dis 2018 Jan 4;111(1):41-52. Epub 2017 Nov 4.

Institut clinique de la Souris, institut de génétique et de biologie moléculaire et cellulaire, université de Strasbourg, Illkirch, France; Laboratoire de neurobiologie et pharmacologie cardiovasculaire, faculté de médecine, fédération de médecine translationnelle, université, CHU de Strasbourg, 11, rue Humann, 67085 Strasbourg cedex, France. Electronic address:

Background: Metabolic syndrome is a combination of symptoms including obesity, dyslipidaemia, glucose intolerance and hypertension. Oxidative stress appears to be a pathophysiological factor that links these signs and encourages progression towards heart failure and diabetes. Nox4 is a hydrogen peroxide nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoform - found in various cardiovascular cells and tissues, but also in tissues such as the liver - which is involved in glucose and lipid homeostasis.

Aims: To test whether inhibition of the Nox4 enzyme could improve blood pressure and metabolic parameters in mice receiving either angiotensin II or a high-fat diet.

Methods: Systolic and diastolic arterial pressures, pulse rate and heart rate were obtained in 24 male mice (12 wild-type [WT] and 12 Nox4) before and during 14 days of angiotensin II infusion. After angiotensin II infusion, cardiac histological remodeling was assessed. Weight and biochemical parameters were measured in 18 male and 18 female mice (nine WT and nine Nox4 per gender) after 10 weeks on a standard chow diet, then 15 weeks on a high-fat diet. Glucose tolerance and insulin sensitivity were tested at age 25 weeks.

Results: Knock-out animals did not demonstrate a baseline blood pressure phenotype, but blocking Nox4 protected against angiotensin II-mediated arterial and pulse pressure increases. No protection against angiotensin II-induced cardiac fibrosis was observed. From a metabolic point of view, Nox4 inhibition reduced plasma triglycerides in male and female mice under a chow diet. However, Nox4 deletion did not affect the metabolic profile under a high-fat diet in males or females, but increased glucose intolerance in females.

Conclusion: Our data identify Nox4 as a key source of radical oxygen species involved in hypertension and some metabolic problems.
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http://dx.doi.org/10.1016/j.acvd.2017.03.011DOI Listing
January 2018

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

PLoS Genet 2017 Jul 13;13(7):e1006886. Epub 2017 Jul 13.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Illkirch, France.

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.
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http://dx.doi.org/10.1371/journal.pgen.1006886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5531616PMC
July 2017

Modeling human disease in rodents by CRISPR/Cas9 genome editing.

Mamm Genome 2017 08 4;28(7-8):291-301. Epub 2017 Jul 4.

CELPHEDIA, PHENOMIN, Institut Clinique de la Souris (ICS), CNRS, INSERM, University of Strasbourg, 1 rue Laurent Fries, 67404, Illkirch, France.

Modeling human disease has proven to be a challenge for the scientific community. For years, generating an animal model was complicated and restricted to very few species. With the rise of CRISPR/Cas9, it is now possible to generate more or less any animal model. In this review, we will show how this technology is and will change our way to obtain relevant disease animal models and how it should impact human health.
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http://dx.doi.org/10.1007/s00335-017-9703-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5569124PMC
August 2017

Deciphering the Role of Oncogenic MITFE318K in Senescence Delay and Melanoma Progression.

J Natl Cancer Inst 2017 08;109(8)

INSERM, U1065 (équipe 1), Equipe labélisée ARC 2016, C3M, Nice, France.

Background: MITF encodes an oncogenic lineage-specific transcription factor in which a germline mutation ( MITFE318K ) was identified in human patients predisposed to both nevus formation and, among other tumor types, melanoma. The molecular mechanisms underlying the oncogenic activity of MITF E318K remained uncharacterized.

Methods: Here, we compared the SUMOylation status of endogenous MITF by proximity ligation assay in melanocytes isolated from wild-type (n = 3) or E318K (n = 4) MITF donors. We also used a newly generated Mitf E318K knock-in (KI) mouse model to assess the role of Mitf E318K (n = 7 to 13 mice per group) in tumor development in vivo and performed transcriptomic analysis of the tumors to identify the molecular mechanisms. Finally, using immortalized or normal melanocytes (wild-type or E318K MITF, n = 2 per group), we assessed the role of MITF E318K on the induction of senescence mediated by BRAF V600E . All statistical tests were two-sided.

Results: We demonstrated a decrease in endogenous MITF SUMOylation in melanocytes from MITF E318K patients (mean of cells with hypoSUMOylated MITF, MITF E318K vs MITF WT , 94% vs 44%, difference = 50%, 95% CI = 21.8% to 67.2%, P  = .004). The Mitf E318K mice were slightly hypopigmented (mean melanin content Mitf WT vs Mitf E318K/+ , 0.54 arbitrary units [AU] vs 0.36 AU, difference = -0.18, 95% CI = -0.36 to -0.007, P  = .04). We provided genetic evidence that Mitf E318K enhances BRaf V600E -induced nevus formation in vivo (mean nevus number for Mitf E318K , BRaf V600E vs Mitf WT , BRaf V600E , 68 vs 44, difference = 24, 95% CI = 9.1 to 38.9, P  = .006). Importantly, although Mitf E318K was not sufficient to cooperate with BRaf V600E alone in promoting metastatic melanoma, it accelerated tumor formation on a BRaf V600E , Pten-deficient background (median survival, Mitf E318K/+  = 42 days, 95% CI = 31 to 46 vs Mitf WT  = 51 days, 95% CI = 50 to 55, P  < .001). Transcriptome analysis suggested a decrease in senescence in tumors from Mitf E318K mice. We confirmed this hypothesis by in vitro experiments, demonstrating that Mitf E318K impaired the ability of human melanocytes to undergo BRAF V600E -induced senescence.

Conclusions: We characterized the functions of melanoma-associated MITF E318K mutations. Our results demonstrate that MITF E318K reduces the program of senescence to potentially favor melanoma progression in vivo.
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http://dx.doi.org/10.1093/jnci/djw340DOI Listing
August 2017

Efficient and rapid generation of large genomic variants in rats and mice using CRISMERE.

Sci Rep 2017 03 7;7:43331. Epub 2017 Mar 7.

PHENOMIN, Institut Clinique de la Souris (ICS), CNRS, INSERM, University of Strasbourg, 1 rue Laurent Fries, F-67404 Illkirch-Graffenstaden, France.

Modelling Down syndrome (DS) in mouse has been crucial for the understanding of the disease and the evaluation of therapeutic targets. Nevertheless, the modelling so far has been limited to the mouse and, even in this model, generating duplication of genomic regions has been labour intensive and time consuming. We developed the CRISpr MEdiated REarrangement (CRISMERE) strategy, which takes advantage of the CRISPR/Cas9 system, to generate most of the desired rearrangements from a single experiment at much lower expenses and in less than 9 months. Deletions, duplications, and inversions of genomic regions as large as 24.4 Mb in rat and mouse founders were observed and germ line transmission was confirmed for fragment as large as 3.6 Mb. Interestingly we have been able to recover duplicated regions from founders in which we only detected deletions. CRISMERE is even more powerful than anticipated it allows the scientific community to manipulate the rodent and probably other genomes in a fast and efficient manner which was not possible before.
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http://dx.doi.org/10.1038/srep43331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339700PMC
March 2017

Translation of Expanded CGG Repeats into FMRpolyG Is Pathogenic and May Contribute to Fragile X Tremor Ataxia Syndrome.

Neuron 2017 Jan 5;93(2):331-347. Epub 2017 Jan 5.

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR7104, University of Strasbourg, 67400 Illkirch, France; Université de Strasbourg, 67000 Strasbourg, France; Centre National de la Recherche Scientifique, UMR7104, 67400 Illkirch, France; Institut National de la Santé et de la Recherche Médicale, U964, 67400 Illkirch, France. Electronic address:

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a limited expansion of CGG repeats in the 5' UTR of FMR1. Two mechanisms are proposed to cause FXTAS: RNA gain-of-function, where CGG RNA sequesters specific proteins, and translation of CGG repeats into a polyglycine-containing protein, FMRpolyG. Here we developed transgenic mice expressing CGG repeat RNA with or without FMRpolyG. Expression of FMRpolyG is pathogenic, while the sole expression of CGG RNA is not. FMRpolyG interacts with the nuclear lamina protein LAP2β and disorganizes the nuclear lamina architecture in neurons differentiated from FXTAS iPS cells. Finally, expression of LAP2β rescues neuronal death induced by FMRpolyG. Overall, these results suggest that translation of expanded CGG repeats into FMRpolyG alters nuclear lamina architecture and drives pathogenesis in FXTAS.
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http://dx.doi.org/10.1016/j.neuron.2016.12.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5263258PMC
January 2017

Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction.

BMC Cell Biol 2016 08 5;17(1):30. Epub 2016 Aug 5.

The Mary Lyon Centre, Medical Research Council Harwell Institute, Harwell Science and Innovation Campus, Didcot, OX11 0RD, Oxon, UK.

Background: Karyotypic integrity is essential for the successful germline transmission of alleles mutated in embryonic stem (ES) cells. Classical methods for the identification of aneuploidy involve cytological analyses that are both time consuming and require rare expertise to identify mouse chromosomes.

Results: As part of the International Mouse Phenotyping Consortium, we gathered data from over 1,500 ES cell clones and found that the germline transmission (GLT) efficiency of clones is compromised when over 50 % of cells harbour chromosome number abnormalities. In JM8 cells, chromosomes 1, 8, 11 or Y displayed copy number variation most frequently, whilst the remainder generally remain unchanged. We developed protocols employing droplet digital polymerase chain reaction (ddPCR) to accurately quantify the copy number of these four chromosomes, allowing efficient triage of ES clones prior to microinjection. We verified that assessments of aneuploidy, and thus decisions regarding the suitability of clones for microinjection, were concordant between classical cytological and ddPCR-based methods. Finally, we improved the method to include assay multiplexing so that two unstable chromosomes are counted simultaneously (and independently) in one reaction, to enhance throughput and further reduce the cost.

Conclusion: We validated a PCR-based method as an alternative to classical karyotype analysis. This technique enables laboratories that are non-specialist, or work with large numbers of clones, to precisely screen ES cells for the most common aneuploidies prior to microinjection to ensure the highest level of germline transmission potential. The application of this method allows early exclusion of aneuploid ES cell clones in the ES cell to mouse conversion process, thus improving the chances of obtaining germline transmission and reducing the number of animals used in failed microinjection attempts. This method can be applied to any other experiments that require accurate analysis of the genome for copy number variation (CNV).
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http://dx.doi.org/10.1186/s12860-016-0108-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974727PMC
August 2016

Counterregulation between thymic stromal lymphopoietin- and IL-23-driven immune axes shapes skin inflammation in mice with epidermal barrier defects.

J Allergy Clin Immunol 2016 07 15;138(1):150-161.e13. Epub 2016 Mar 15.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique UMR7104/Institut National de la Santé et de la Recherche Médicale U964/Université de Strasbourg, Illkirch, France; University of Strasbourg Institute for Advanced Study, Strasbourg, France; Freiburg Institute for Advanced Studies, Freiburg, Germany. Electronic address:

Background: Epidermal barrier dysfunction has been recognized as a critical factor in the initiation and exacerbation of skin inflammation, particularly in patients with atopic dermatitis (AD) and AD-like congenital disorders, including peeling skin syndrome type B. However, inflammatory responses developed in barrier-defective skin, as well as the underlying mechanisms, remained incompletely understood.

Objective: We aimed to decipher inflammatory axes and the cytokine network in mouse skin on breakdown of epidermal stratum corneum barrier.

Methods: We generated Cdsn(iep-/-) mice with corneodesmosin ablation in keratinocytes selectively in an inducible manner. We characterized inflammatory responses and cytokine expression by using histology, immunohistochemistry, ELISA, and quantitative PCR. We combined mouse genetic tools, antibody-mediated neutralization, signal-blocking reagents, and topical antibiotic treatment to explore the inflammatory axes.

Results: We show that on breakdown of the epidermal stratum corneum barrier, type 2 and type 17 inflammatory responses are developed simultaneously, driven by thymic stromal lymphopoietin (TSLP) and IL-23, respectively. Importantly, we reveal a counterregulation between these 2 inflammatory axes. Furthermore, we show that protease-activated receptor 2 signaling is involved in mediating the TSLP/type 2 axis, whereas skin bacteria are engaged in induction of the IL-23/type 17 axis. Moreover, we find that IL-1β is induced in skin of Cdsn(iep-/-) mice and that blockade of IL-1 signaling suppresses both TSLP and IL-23 expression and ameliorates skin inflammation.

Conclusion: The inflammatory phenotype in barrier-defective skin is shaped by counterregulation between the TSLP/type 2 and IL-23/type 17 axes. Targeting IL-1 signaling could be a promising therapeutic option for controlling skin inflammation in patients with peeling skin syndrome type B and other diseases related to epidermal barrier dysfunction, including AD.
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http://dx.doi.org/10.1016/j.jaci.2016.01.013DOI Listing
July 2016

Toxic gain of function from mutant FUS protein is crucial to trigger cell autonomous motor neuron loss.

EMBO J 2016 05 7;35(10):1077-97. Epub 2016 Mar 7.

Faculté de Médecine, INSERM U1118, Strasbourg, France Université de Strasbourg UMR_S1118, Strasbourg, France

FUS is an RNA-binding protein involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS-containing aggregates are often associated with concomitant loss of nuclear FUS Whether loss of nuclear FUS function, gain of a cytoplasmic function, or a combination of both lead to neurodegeneration remains elusive. To address this question, we generated knockin mice expressing mislocalized cytoplasmic FUS and complete FUS knockout mice. Both mouse models display similar perinatal lethality with respiratory insufficiency, reduced body weight and length, and largely similar alterations in gene expression and mRNA splicing patterns, indicating that mislocalized FUS results in loss of its normal function. However, FUS knockin mice, but not FUS knockout mice, display reduced motor neuron numbers at birth, associated with enhanced motor neuron apoptosis, which can be rescued by cell-specific CRE-mediated expression of wild-type FUS within motor neurons. Together, our findings indicate that cytoplasmic FUS mislocalization not only leads to nuclear loss of function, but also triggers motor neuron death through a toxic gain of function within motor neurons.
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http://dx.doi.org/10.15252/embj.201592559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4868956PMC
May 2016

The cell proliferation antigen Ki-67 organises heterochromatin.

Elife 2016 Mar 7;5:e13722. Epub 2016 Mar 7.

Montpellier Institute of Molecular Genetics (IGMM) CNRS UMR 5535, Centre National de la Recherche Scientifique (CNRS), Montpellier, France.

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.
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http://dx.doi.org/10.7554/eLife.13722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4841783PMC
March 2016

Reciprocal Effects on Neurocognitive and Metabolic Phenotypes in Mouse Models of 16p11.2 Deletion and Duplication Syndromes.

PLoS Genet 2016 Feb 12;12(2):e1005709. Epub 2016 Feb 12.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.

The 16p11.2 600 kb BP4-BP5 deletion and duplication syndromes have been associated with developmental delay; autism spectrum disorders; and reciprocal effects on the body mass index, head circumference and brain volumes. Here, we explored these relationships using novel engineered mouse models carrying a deletion (Del/+) or a duplication (Dup/+) of the Sult1a1-Spn region homologous to the human 16p11.2 BP4-BP5 locus. On a C57BL/6N inbred genetic background, Del/+ mice exhibited reduced weight and impaired adipogenesis, hyperactivity, repetitive behaviors, and recognition memory deficits. In contrast, Dup/+ mice showed largely opposite phenotypes. On a F1 C57BL/6N × C3B hybrid genetic background, we also observed alterations in social interaction in the Del/+ and the Dup/+ animals, with other robust phenotypes affecting recognition memory and weight. To explore the dosage effect of the 16p11.2 genes on metabolism, Del/+ and Dup/+ models were challenged with high fat and high sugar diet, which revealed opposite energy imbalance. Transcriptomic analysis revealed that the majority of the genes located in the Sult1a1-Spn region were sensitive to dosage with a major effect on several pathways associated with neurocognitive and metabolic phenotypes. Whereas the behavioral consequence of the 16p11 region genetic dosage was similar in mice and humans with activity and memory alterations, the metabolic defects were opposite: adult Del/+ mice are lean in comparison to the human obese phenotype and the Dup/+ mice are overweight in comparison to the human underweight phenotype. Together, these data indicate that the dosage imbalance at the 16p11.2 locus perturbs the expression of modifiers outside the CNV that can modulate the penetrance, expressivity and direction of effects in both humans and mice.
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http://dx.doi.org/10.1371/journal.pgen.1005709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4752317PMC
February 2016

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

Hum Mol Genet 2015 Dec 16;24(23):6736-55. Epub 2015 Sep 16.

Institut Clinique de la Souris, PHENOMIN, GIE CERBM, 1 rue Laurent Fries, 67404 Illkirch, France, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France, Centre National de la Recherche Scientifique, UMR7104, Illkirch, France, Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France and Université de Strasbourg, Illkirch, France

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 (Atp6ap2(Camk2aCre/0) mice) caused increased spontaneous locomotor activity and altered fear memory. Both Drosophila ATP6AP2 knockdown and Atp6ap2(Camk2aCre/0) mice presented with presynaptic transmission defects, and with an abnormal number and morphology of synapses. In addition, Atp6ap2(Camk2aCre/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 Atp6ap2(Camk2aCre/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.
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http://dx.doi.org/10.1093/hmg/ddv380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4634377PMC
December 2015

Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics.

Authors:
Martin Hrabě de Angelis George Nicholson Mohammed Selloum Jacqui White Hugh Morgan Ramiro Ramirez-Solis Tania Sorg Sara Wells Helmut Fuchs Martin Fray David J Adams Niels C Adams Thure Adler Antonio Aguilar-Pimentel Dalila Ali-Hadji Gregory Amann Philippe André Sarah Atkins Aurelie Auburtin Abdel Ayadi Julien Becker Lore Becker Elodie Bedu Raffi Bekeredjian Marie-Christine Birling Andrew Blake Joanna Bottomley Mike Bowl Véronique Brault Dirk H Busch James N Bussell Julia Calzada-Wack Heather Cater Marie-France Champy Philippe Charles Claire Chevalier Francesco Chiani Gemma F Codner Roy Combe Roger Cox Emilie Dalloneau André Dierich Armida Di Fenza Brendan Doe Arnaud Duchon Oliver Eickelberg Chris T Esapa Lahcen El Fertak Tanja Feigel Irina Emelyanova Jeanne Estabel Jack Favor Ann Flenniken Alessia Gambadoro Lilian Garrett Hilary Gates Anna-Karin Gerdin George Gkoutos Simon Greenaway Lisa Glasl Patrice Goetz Isabelle Goncalves Da Cruz Alexander Götz Jochen Graw Alain Guimond Wolfgang Hans Geoff Hicks Sabine M Hölter Heinz Höfler John M Hancock Robert Hoehndorf Tertius Hough Richard Houghton Anja Hurt Boris Ivandic Hughes Jacobs Sylvie Jacquot Nora Jones Natasha A Karp Hugo A Katus Sharon Kitchen Tanja Klein-Rodewald Martin Klingenspor Thomas Klopstock Valerie Lalanne Sophie Leblanc Christoph Lengger Elise le Marchand Tonia Ludwig Aline Lux Colin McKerlie Holger Maier Jean-Louis Mandel Susan Marschall Manuel Mark David G Melvin Hamid Meziane Kateryna Micklich Christophe Mittelhauser Laurent Monassier David Moulaert Stéphanie Muller Beatrix Naton Frauke Neff Patrick M Nolan Lauryl Mj Nutter Markus Ollert Guillaume Pavlovic Natalia S Pellegata Emilie Peter Benoit Petit-Demoulière Amanda Pickard Christine Podrini Paul Potter Laurent Pouilly Oliver Puk David Richardson Stephane Rousseau Leticia Quintanilla-Fend Mohamed M Quwailid Ildiko Racz Birgit Rathkolb Fabrice Riet Janet Rossant Michel Roux Jan Rozman Ed Ryder Jennifer Salisbury Luis Santos Karl-Heinz Schäble Evelyn Schiller Anja Schrewe Holger Schulz Ralf Steinkamp Michelle Simon Michelle Stewart Claudia Stöger Tobias Stöger Minxuan Sun David Sunter Lydia Teboul Isabelle Tilly Glauco P Tocchini-Valentini Monica Tost Irina Treise Laurent Vasseur Emilie Velot Daniela Vogt-Weisenhorn Christelle Wagner Alison Walling Bruno Weber Olivia Wendling Henrik Westerberg Monja Willershäuser Eckhard Wolf Anne Wolter Joe Wood Wolfgang Wurst Ali Önder Yildirim Ramona Zeh Andreas Zimmer Annemarie Zimprich Chris Holmes Karen P Steel Yann Herault Valérie Gailus-Durner Ann-Marie Mallon Steve Dm Brown

Nat Genet 2015 Sep 27;47(9):969-978. Epub 2015 Jul 27.

MRC Harwell, Medical Research Council, Harwell, UK.

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.
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http://dx.doi.org/10.1038/ng.3360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564951PMC
September 2015

Rfx6 maintains the functional identity of adult pancreatic β cells.

Cell Rep 2014 Dec 11;9(6):2219-32. Epub 2014 Dec 11.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Institut National de la Santé et de la Recherche Médicale U964, Centre National de Recherche Scientifique UMR7104, Université de Strasbourg, Illkirch 67404, France. Electronic address:

Increasing evidence suggests that loss of β cell characteristics may cause insulin secretory deficiency in diabetes, but the underlying mechanisms remain unclear. Here, we show that Rfx6, whose mutation leads to neonatal diabetes in humans, is essential to maintain key features of functionally mature β cells in mice. Rfx6 loss in adult β cells leads to glucose intolerance, impaired β cell glucose sensing, and defective insulin secretion. This is associated with reduced expression of core components of the insulin secretion pathway, including glucokinase, the Abcc8/SUR1 subunit of KATP channels and voltage-gated Ca(2+) channels, which are direct targets of Rfx6. Moreover, Rfx6 contributes to the silencing of the vast majority of "disallowed" genes, a group usually specifically repressed in adult β cells, and thus to the maintenance of β cell maturity. These findings raise the possibility that changes in Rfx6 expression or activity may contribute to β cell failure in humans.
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http://dx.doi.org/10.1016/j.celrep.2014.11.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4542305PMC
December 2014

Hyperactivation of Alk induces neonatal lethality in knock-in AlkF1178L mice.

Oncotarget 2014 May;5(9):2703-13

Inserm U830, Paris, France;

The ALK (Anaplastic Lymphoma Kinase) gene encodes a tyrosine kinase receptor preferentially expressed in the central and peripheral nervous systems. A syndromic presentation associating congenital neuroblastoma with severe encephalopathy and an abnormal shape of the brainstem has been described in patients harbouring de novo germline F1174V and F1245V ALK mutations. Here, we investigated the phenotype of knock-in (KI) mice bearing the AlkF1178L mutation (F1174L in human). Although heterozygous KI mice did not reproduce the severe breathing and feeding difficulties observed in human patients, behavioral tests documented a reduced activity during dark phases and an increased anxiety of mutated mice. Matings of heterozygotes yielded the expected proportions of wild-type, heterozygotes and homozygotes at birth but a high neonatal lethality was noticed for homozygotes. We documented Alk expression in several motor nuclei of the brainstem involved in the control of sucking and swallowing. Evaluation of basic physiological functions 12 hours after birth revealed slightly more apneas but a dramatic reduced milk intake for homozygotes compared to control littermates. Overall, our data demonstrate that Alk activation above a critical threshold is not compatible with survival in mice, in agreement with the extremely severe phenotype of patients carrying aggressive de novo ALK germline mutations.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4058038PMC
http://dx.doi.org/10.18632/oncotarget.1882DOI Listing
May 2014