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Genome editing tools have already revolutionized biomedical research and are also expected to have an important impact in the clinic. However, their extensive use in research has revealed much unpredictability, both off and on target, in the outcome of their application. We discuss the challenges associated with this unpredictability, both for research and in the clinic. For the former, an extensive validation of the model is essential. For the latter, potential unpredicted activity does not preclude the use of these tools but requires that molecular evidence to underpin the relevant risk:benefit evaluation is available. Safe and successful clinical application will also depend on the mode of delivery and the cellular context., Graphical Abstract, Genome editing tools have already revolutionized biomedical research and will have a major impact in the clinic. However, the unpredictability of genome editing outcomes raises the question of its safety for human therapy. This article reviews molecular evidences both in animals models and human and their implications for risk:benefit evaluation.

The French mouse clinic (Institut Clinique de la Souris; ICS) has produced more than 2000 targeting vectors for ‘à la carte’ mutagenesis in C57BL/6N mice. Although most of the vectors were used successfully for homologous recombination in murine embryonic stem cells (ESCs), a few have failed to target a specific locus after several attempts. We show here that co-electroporation of a CRISPR plasmid with the same targeting construct as the one that failed previously allows the systematic achievement of positive clones. A careful validation of these clones is, however, necessary as a significant number of clones (but not all) show a concatemerization of the targeting plasmid at the locus. A detailed Southern blot analysis permitted characterization of the nature of these events as standard long-range 5′ and 3′ PCRs were not able to distinguish between correct and incorrect alleles. We show that a simple and inexpensive PCR performed prior to ESC amplification allows detection and elimination of those clones with concatemers. Finally, although we only tested murine ESCs, our results highlight the risk of mis-validation of any genetically modified cell line (such as established lines, induced pluripotent stem cells or those used for ex vivo gene therapy) that combines the use of CRISPR/Cas9 and a circular double-stranded donor. We strongly advise the CRISPR community to perform a Southern blot with internal probes when using CRISPR to enhance homologous recombination in any cell type, including fertilized oocytes.

Perturbation of the excitation/inhibition (E/I) balance leads to neurodevelopmental diseases including to autism spectrum disorders, intellectual disability, and epilepsy. Loss-of-function mutations in the DYRK1A gene, located on human chromosome 21 (Hsa21,) lead to an intellectual disability syndrome associated with microcephaly, epilepsy, and autistic troubles. Overexpression of DYRK1A, on the other hand, has been linked with learning and memory defects observed in people with Down syndrome (DS). Dyrk1a is expressed in both glutamatergic and GABAergic neurons, but its impact on each neuronal population has not yet been elucidated. Here we investigated the impact of Dyrk1a gene copy number variation in glutamatergic neurons using a conditional knockout allele of Dyrk1a crossed with the Tg(Camk2-Cre)4Gsc transgenic mouse. We explored this genetic modification in homozygotes, heterozygotes and combined with the Dp(16Lipi-Zbtb21)1Yey trisomic mouse model to unravel the consequence of Dyrk1a dosage from 0 to 3, to understand its role in normal physiology, and in MRD7 and DS. Overall, Dyrk1a dosage in postnatal glutamatergic neurons did not impact locomotor activity, working memory or epileptic susceptibility, but revealed that Dyrk1a is involved in long-term explicit memory. Molecular analyses pointed at a deregulation of transcriptional activity through immediate early genes and a role of DYRK1A at the glutamatergic post-synapse by deregulating and interacting with key post-synaptic proteins implicated in mechanism leading to long-term enhanced synaptic plasticity. Altogether, our work gives important information to understand the action of DYRK1A inhibitors and have a better therapeutic approach., Author summary The Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A, DYRK1A, drives cognitive alterations with increased dose in Down syndrome (DS) or with reduced dose in DYRK1A-related intellectual disability syndromes (ORPHA:268261; ORPHA:464311) also known as mental retardation, autosomal dominant disease 7 (MRD7; OMIM #614104). Here we report that specific and complete loss of Dyrk1a in glutamatergic neurons induced a range of specific cognitive phenotypes and alter the expression of genes involved in neurotransmission in the hippocampus. We further explored the consequences of Dyrk1a dosage in glutamatergic neurons on the cognitive phenotypes observed respectively in MRD7 and DS mouse models and we found specific roles in long-term explicit memory with no impact on motor activity, short-term working memory, and susceptibility to epilepsy. Then we demonstrated that DYRK1A is a component of the glutamatergic post-synapse and interacts with several component such as NR2B and PSD95. Altogether our work describes a new role of DYRK1A at the glutamatergic synapse that must be considered to understand the consequence of treatment targeting DYRK1A in disease.

International audience; 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. Indepth 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 knockout , 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 knockout 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 T2 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.

Genetic factors are known to contribute to Late Onset Alzheimer’s disease (LOAD) but their contribution to pathophysiology, specially to prodomic phases accessible to therapeutic approaches are far to be understood. To translate genetic risk of Alzheimer's disease (AD) into mechanistic insight, we generated transgenic mouse lines that express a ~195 kbp human BAC that includes only BIN1, a gene associated to LOAD. This model gives a modest BIN1 overexpression, dependent of the number of BAC copies. At 6 months of age, we detected impaired entorhinal cortex (EC)-hippocampal pathways with specific impairments in EC-dentate gyrus synaptic long-term potentiation, dendritic spines of granular cells and recognition episodic memory. Structural changes were quantified using MRI. Their whole-brain functional impact were analyzed using resting state fMRI with a hypoconnectivity centered on entorhinal cortex. These early phenotype defects independent of any changes in A-beta can be instrumental in the search for new AD drug targets.

The reproducibility of research using laboratory animals requires reliable management of their quality, in particular of their genetics, health and environment, all of which contribute to their phenotypes. The point at which these biological materials are transferred between researchers is particularly sensitive, as it may result in a loss of integrity of the animals and/or their documentation. Here, we describe the various aspects of laboratory animal quality that should be confirmed when sharing rodent research models. We also discuss how repositories of biological materials support the scientific community to ensure the continuity of the quality of laboratory animals. Both the concept of quality and the role of repositories themselves extend to all exchanges of biological materials and all networks that support the sharing of these reagents.

International audience; 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.

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.

Fructose consumption in humans and animals has been linked to enhanced de novo lipogenesis, dyslipidemia, and insulin resistance. Hereditary deficiency of ketohexokinase (KHK), the first enzymatic step in fructose metabolism, leads to essential fructosuria in humans, characterized by elevated levels of blood and urinary fructose following fructose ingestion but is otherwise clinically benign. To address whether KHK deficiency is associated with altered glucose and lipid metabolism, a Khk knockout (KO) mouse line was generated and characterized. NMR spectroscopic analysis of plasma following ingestion of [6-13C] fructose revealed striking differences in biomarkers of fructose metabolism. Significantly elevated urine and plasma 13C-fructose levels were observed in Khk KO vs. wild-type (WT) control mice, as was reduced conversion of 13C-fructose into plasma 13C-glucose and 13C-lactate. In addition, the observation of significant levels of fructose-6-phosphate in skeletal muscle tissue of Khk KO, but not WT, mice suggests a potential mechanism, whereby fructose is metabolized via muscle hexokinase in the absence of KHK. Khk KO mice on a standard chow diet displayed no metabolic abnormalities with respect to ambient glucose, glucose tolerance, body weight, food intake, and circulating trigylcerides, β-hydroxybutyrate, and lactate. When placed on a high-fat and high-fructose (HF/HFruc) diet, Khk KO mice had markedly reduced liver weight, triglyceride levels, and insulin levels. Together, these results suggest that Khk KO mice may serve as a good model for essential fructosuria in humans and that inhibition of KHK offers the potential to protect from diet-induced hepatic steatosis and insulin resistance.

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.

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 Tubg1Y92C/+ mouse model and assessed consequences of the mutation. Although centrosomal positioning in bipolar neurons is correct, they fail to initiate locomotion. Furthermore, Tubg1Y92C/+ animals show neuroanatomical and behavioral defects and increased epileptic cortical activity. We show that Tubg1Y92C/+ mice partially mimic the human phenotype and therefore represent a relevant model for further investigations of the physiopathology of cortical malformations., New mutations and genes associated with malformations of cortical development keep being identified, yet there is little known about the underlying cellular mechanisms controlling these impairments. Here, authors generate and characterize a heterozygous TUBG1 knock-in mouse model bearing one of these known mutations and show that TUBG1 mutation leads to the miss-positioning of neurons in the cortical wall due to migration, because of defective microtubules dynamics, and not proliferation defects during corticogenesis.

Genotyping consists of searching for a DNA sequence variation localized at a well-defined locus in the genome. It is an essential step in animal research because it allows the identification of animals that will be bred to generate and maintain a colony, euthanized to control the available space in the animal facility, or used in experiment protocols. Here we describe polymerase chain reaction (PCR) genotyping protocols for fast, sensitive, easy, and cost-effective characterization of mouse genotype. We discuss optimization of parameters to improve the reliability of each assay and propose recommendations for enhancing reproducibility and reducing the occurrence of inconclusive genotyping. All steps required for efficient genotyping are presented: tissue collection; sample verification and direct DNA lysis; establishment of a robust genotyping strategy with reliable, rapid, and cost-effective assays; and finally, transition to high-throughput automatized PCR, including mix miniaturization and automation. © 2019 The Authors. Basic Protocol 1: Tissue sampling methods and procedure Basic Protocol 2: Sample verification and DNA lysis Basic Protocol 3: Design of a genotyping strategy Basic Protocol 4: Moving to high-throughput genotyping.

Late Onset Alzheimer Disease (LOAD) is the most common form of dementia and one of the most challenging diseases of modern society. Understanding the preclinical stages of AD that begins in the brain at least 2 to 3 decades before evidence of episodic memory defects in patients is pivotal for the design of successful approaches to delay or reverse the transition from normal brain function to cognitive impairments. Our working hypothesis is that LOAD genetic risk factors can be sufficient to generate early phenotypical changes before any changes in either Abeta or Tau. To test this hypothesis, we generated an hBIN1 mouse model based on the human BIN1 gene overexpression that we found in post-mortem brain samples from LOAD patients. BIN1 is the second important risk factor for AD, following the APOE gene. We identified co-deregulated gene repertoires common to both 7-week mouse hippocampus sub-regions and post-mortem brain samples from LOAD patients, demonstrating the validity of this hBIN1 model. We evidenced an early phenotype of neurodegeneration starting at 3 months with structural impairment fiber pathways quantified by high resolution (17.2T) (MRI DTI) and related functional impacts. We found structural changes in entorhinal cortex-dentate gyrus (EC-DG) pathway known to be the earliest brain region impacted in LOAD. Similarly, the function of this pathway was impaired both in vitro and in vivo, with the changes in spine density and dendritic simplification of DG neurons, impaired EC-DG long-term potentiation (LTP) and behavioral deficits linked to object recognition episodic memory. As expected for a neurodegenerative model, we evidenced the progression of dysfunction at the morphological, functional and behavioral levels with age. Structural spreading involved impairment of fibers in somatosensory and temporal associative cortexes at month 15. Functional and behavioral spreading was characterized by impact on pattern separation of spatial episodic memory. Moreover, this neurodegeneration occurred without any detectable changes in Abeta 1-42 and tau. Overall, these data show the possibility to identify a repertoire of molecular changes occurring both in patients and in hBIN1 mice and whose further manipulation can be instrumental to rescue or delay episodic memory defects.

Novel development in mouse phenotyping 2014: et al., The International Knockout Mouse Consortium (IKMC) developed high throughput gene trapping and gene targeting pipelines that produced mostly conditional mutations of more than 18,500 genes in C57BL/6N mouse embryonic stem (ES) cells which have been archived and are freely available to the research community as a frozen resource. From this unprecedented resource more than 6000 mutant mouse strains have been generated by the IKMC in collaboration with the International Mouse Phenotyping Consortium (IMPC). In addition, a cre-driver resource was established including 250 C57BL/6 cre-inducible mouse strains. Complementing the cre-driver resource, a collection comprising 27 rAAVs expressing cre in a tissue-specific manner has also been produced. All resources are easily accessible from the IKMC/IMPC web portal (www.mousephenotype.org). The IKMC/IMPC resource is a standardized reference library of mouse models with defined genetic backgrounds enabling the analysis of gene-disease associations in mice of different genetic makeup and should therefore have a major impact on biomedical research., The authors are supported by the EUCOMMTOOLS project which is funded by the European Commission [FP7-HEALTH-F4-2010-261492] and UM1-HG006370-06 (TFM, JW); the National Insitute of Health U54 HG006370 (TFM, DS and SDMB), U42 OD011185 (SAM), HG006364-03S1 (KCKL), and U42 OD011175 (CM and KCKL); NorCommTLS (MRI, Government of Ontario) and Genome Canada (OG-090) (LMJN, CM); the Manitoba Research Innovation Fund (GGH); Genome British Columbia AGCP-CanEuCre-01 award (EMS). National Centre for Scientific Research (CNRS), the French National Institute of Health and Medical Research (INSERM), the University of Strasbourg (UDS), the “Centre Européen de Recherche en Biologie et en Médecine” the French state funds through the “Agence Nationale de la Recherche”, Investissements d’Avenir labelled ANR-10-IDEX-0002-02, ANR-10-LABX-0030-INRT, ANR-10-INBS-07 PHENOMIN to YH.

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.

Summary 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., Highlights • CGG repeats in the 5′ UTR of FMR1 are translated through initiation to an ACG codon • Translation of CGG repeats in the polyglycine protein, FMRpolyG, is toxic in mice • FMRpolyG binds and disrupts protein of the nuclear lamina, Sellier et al. show that translation of expanded CGG repeats located in the 5′ UTR of the FMR1 gene require an upstream ACG near-cognate initiation codon. Translation of CGG repeats into a short polyglycine-containing protein, FMRpolyG, is pathogenic in mouse models.

Summary 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.

ATP6AP2 codes for the (pro)renin receptor and is an essential component of vacuolar H+ ATPase. Activating (pro)renin for conversion of Angiotensinogen to Angiotensin makes ATP6AP2 attractive for drug intervention. Tissue-specific ATP6AP2 inactivation in mouse suggested a strong impact on various organs. Consistent with this, we found that embryonic ablation of Atp6ap2 resulted in both male hemizygous lethality and female haploinsufficiency. Next, we examined the phenotype of an induced inactivation in the adult animal, most akin to detect potential effect of functional interference of ATP6AP2 through drug therapy. Induced ablation of Atp6ap2, even without equal efficiency in all tissues (aorta, brain and kidney), resulted in rapid lethality marked by weight loss, changes in nutritional as well as blood parameters, leukocyte depletion, and bone marrow hypoplasia. Upon Atp6ap2 ablation, the colon demonstrated a rapid disruption of crypt morphology, aberrant proliferation, cell-death activation, as well as generation of microadenomas. Consequently, disruption of ATP6AP2 is extremely poorly tolerated in the adult, and severely affects various organ systems demonstrating that ATP6AP2 is an essential gene implicated in basic cellular mechanisms and necessary for multiple organ function. Accordingly, any potential drug targeting of this gene product must be strictly assessed for safety.

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.

Fibroblasts constitute the major mesenchymal cell type in the connective tissue and their functions are remarkably diverse: here, by characterising lineages of mouse skin fibroblasts, it is shown that distinct subpopulations contribute to skin development and repair during injury. Fibroblasts are unremarkable looking cells found in most tissues in the body, where they are mainly concerned with making the collagen that supports other cell types. The cells all look much the same yet are functionally diverse, prompting the question, is there just one cell type responding differently to different stimuli, or do individual cells specialize? A transplantation and lineage tracing study in mice now shows that skin connective tissue arises from two distinct fibroblast lineages that also contribute differentially to skin development and repair after injury. One cell type forms the lower dermis and the other the upper dermis. The latter lineage is required for hair follicle production. In wounded adult skin, the initial wave of dermal repair is mediated by the 'lower' lineage, which may explain the absence of hair follicles in newly closed wounds. The authors develop a comprehensive lineage tree for all fibroblast-derived cell types in mouse dermis, including smooth muscle cells and adipocytes. Fibroblasts are the major mesenchymal cell type in connective tissue and deposit the collagen and elastic fibres of the extracellular matrix (ECM)1. Even within a single tissue, fibroblasts exhibit considerable functional diversity, but it is not known whether this reflects the existence of a differentiation hierarchy or is a response to different environmental factors. Here we show, using transplantation assays and lineage tracing in mice, that the fibroblasts of skin connective tissue arise from two distinct lineages. One forms the upper dermis, including the dermal papilla that regulates hair growth and the arrector pili muscle, which controls piloerection. The other forms the lower dermis, including the reticular fibroblasts that synthesize the bulk of the fibrillar ECM, and the preadipocytes and adipocytes of the hypodermis. The upper lineage is required for hair follicle formation. In wounded adult skin, the initial wave of dermal repair is mediated by the lower lineage and upper dermal fibroblasts are recruited only during re-epithelialization. Epidermal β-catenin activation stimulates the expansion of the upper dermal lineage, rendering wounds permissive for hair follicle formation. Our findings explain why wounding is linked to formation of ECM-rich scar tissue that lacks hair follicles2,3,4. They also form a platform for discovering fibroblast lineages in other tissues and for examining fibroblast changes in ageing and disease.

International audience; Mutations of the AMP-activated kinase gamma 2 subunit (AMPKγ2), N488I (AMPKγ2NI) and R531G (AMPKγ2RG), are associated with Wolff-Parkinson-White (WPW) syndrome, a cardiac disorder characterized by ventricular pre-excitation in humans. Cardiac-specific transgenic overexpression of human AMPKγ2NI or AMPKγ2RG leads to constitutive AMPK activation and the WPW phenotype in mice. However, overexpression of these mutant proteins also caused profound, non-physiological increase in cardiac glycogen, which might abnormally alter the true phenotype. To investigate whether physiological levels of AMPKγ2NI or AMPKγ2RG mutation cause WPW syndrome and metabolic changes in other organs, we generated two knock-in mouse lines on the C57BL/6N background harboring mutations of human AMPKγ2NI and AMPKγ2RG, respectively. Similar to the reported phenotypes of mice overexpressing AMPKγ2NI or AMPKγ2RG in the heart, both lines developed WPW syndrome and cardiac hypertrophy; however, these effects were independent of cardiac glycogen accumulation. Compared with AMPKγ2WT mice, AMPKγ2NI and AMPKγ2RG mice exhibited reduced body weight, fat mass, and liver steatosis when fed with a high fat diet (HFD). Surprisingly, AMPKγ2RG but not AMPKγ2NI mice fed with an HFD exhibited severe kidney injury characterized by glycogen accumulation, inflammation, apoptosis, cyst formation, and impaired renal function. These results demonstrate that expression of AMPKγ2NI and AMPKγ2RG mutations at physiological levels can induce beneficial metabolic effects but that this is accompanied by WPW syndrome. Our data also reveal an unexpected effect of AMPKγ2RG in the kidney, linking lifelong constitutive activation of AMPK to a potential risk for kidney dysfunction in the context of an HFD.

To facilitate the use of the new mutant resource developed in the mouse, we have generated Cre and FlpO deleter mice on a pure inbred C57BL/6N background. The new transgenic constructs were designed to drive either the Cre or FlpO recombinase, fused to a specific fluorescent marker, respectively the eGFP or the eYFP, and were inserted by homologous recombination in the neutral Rosa26 locus. They allow a rapid, cost-effective, and efficient identification of the carrier individuals through the coexpression of the fluorescent marker. The recombination efficiency of the two deleter lines, Gt(ROSA)26S ortm1(ACTB-cre,-EGFP)Icsand Gt(ROSA) 26S ortm2(CAG-flpo, EYFP)Ics, was carefully evaluated using five loxP-flanked or four FRT-flanked alleles located at different positions in the mouse genome. For each tested locus, we observed a 100% excision rate. The transgenic mice are easily distinguishable from wild type animals by their bright fluorescence that remains easily detectable until 10 days after birth. In the adult, fluorescence can still be detected in the unpigmented paws. Furthermore, they both display accumulation of the specific recombinase during oogenesis. These fluorescent 'Cre- and Flp- deleter' transgenic lines are valuable tools for the scientific community by their high and stable recombination efficiency, the simplicity of genotype identification and the maintenance of a pure genetic background when used to remove specific selection cassette or to induce complete loss-of-function allele.

Structure et evolution d'une famille d'elements integratifs potentiellement conjugatifs ou mobilisables de Streptococcus thermophilus. Les elements integratifs conjugatifs (ICEs) s'excisent par recombinaison site-specifique sous forme circulaire, se transferent par conjugaison puis s'integrent dans la bacterie receptrice. L'element ICESt1 de Streptococcus thermophilus CNRZ368 (34,7 kb) s'integre et s'excise par recombinaison site-specifique. Par ailleurs, il code un systeme de conjugaison putatif apparente de facon lointaine a eelui de Tn916 d'Enterococcus faecalis et constitue donc un ICE putatif. Quatre elements apparentes a ICESt1 sont integres au meme site que lui chez sept souches de S. thermophilus. L'un des elements, ICESt3 est probablement un ICE. Les trois autres (CIMEs) deriveraient d'un ICE par deletion des modules de conjugaison et de recombinaison. Ces elements codent des fonctions non impliquees dans le transfert comme des systemes de restriction-modification. Chacun d'entre eux presente une structure chimerique resultant de l'acquisition de modules d'origines differentes. L'analyse de leur sequence indique qu'ils sont impliques dans des transferts horizontaux entre S. thermophilus et diverses especes de bacteries lactiques alimentaires ou pathogenes. ACIME308 a echange des modules de restriction-modification et de resistance aux ions Cd ++ avec des plasmides. CIME19258 a acquis un module de resistance aux ions Cd ++ par integration d'un ICE apparente a Tn916 dans un CIME. La recombinaison site-specifique entre un site interne d'ICESt1 de type attL et le site attR provoque l'excision de la partie droite d'ICESt1 qui constituerait elle-meme un ICE, ICESt2. ICESt1 serait ainsi apparu par accretion d'un CIME et d'ICESt2. CIME302, ICESt2 et ICESt3 seraient egalement apparus par accretion site-specifique d'ICEs et de CIMEs et/ou mobilisation de CIMEs par des ICEs. Un ICE s'integrerait par recombinaison site-specifique dans le site attR d'un CIME formant ainsi un element composite (CIME-ICE) par accretion. Celui-ci s'exciserait par recombinaison site-specifique puis se transfererait par conjugaison.

Elements that excise and integrate, such as prophages, and transfer by conjugation, such as plasmids, have been found in various bacteria. These elements appear to have a diversified set of characteristics including cell-to-cell contact using pili or cell aggregation, transfer of single-stranded or double-stranded DNA, low or high specificity of integration and serine or tyrosine recombinases. This has led to a highly heterogeneous nomenclature, including conjugative transposons, integrative 'plasmids', genomic islands and numerous unclassified elements. However, all these elements excise by site-specific recombination, transfer the resulting circular form by conjugation and integrate by recombination between a specific site of this circular form and a site in the genome of their host. Whereas replication of the circular form probably occurs during conjugation, this replication is not involved in the maintenance of the element. In this review, we show that these elements share very similar characteristics and, therefore, we propose to classify them as integrative and conjugative elements (ICEs). These elements evolve by acquisition or exchanges of modules with various transferable elements including at least ICEs and plasmids. The ICEs are probably widespread among the bacteria.

The origin of the cells that contribute to skin fibrosis is unclear. We undertook the present study to assess the contribution of Sox2-expressing skin progenitor cells to bleomycin-induced scleroderma.Scleroderma was induced, by bleomycin administration, in wild-type mice and in mice in which CCN2 was deleted from Sox2-expressing cells. Lineage tracing analysis was performed to assess whether cells expressing Sox2 are recruited to fibrotic lesions in response to bleomycin-induced scleroderma.In response to bleomycin, Sox2-positive/α-smooth muscle actin-positive cells were recruited to fibrotic tissue. CCN2-conditional knockout mice in which CCN2 was deleted from Sox2-expressing cells exhibited resistance to bleomycin-induced skin fibrosis. Collectively, these results indicate that CCN2 is required for the recruitment of progenitor cells and that CCN2-expressing progenitor cells are essential for bleomycin-induced skin fibrosis. Lineage tracing analysis using mice in which a tamoxifen-dependent Cre recombinase was expressed under the control of the Sox2 promoter confirmed that progenitor cells were recruited to the fibrotic lesion in response to bleomycin, and that this did not occur in CCN2-knockout mice. The ability of serum to induce α-smooth muscle actin expression in skin progenitor cells required the presence of CCN2.Sox2-positive skin progenitor cells are required in order for bleomycin-induced skin fibrosis to occur, and CCN2 is required for the recruitment of these cells to the fibrotic lesion. Targeting stem cell recruitment or CCN2 may therefore represent a useful therapeutic approach in combating fibrotic skin disease.

Vesicular (v)- and target (t)-SNARE proteins assemble in SNARE complex to mediate membrane fusion. Tetanus neurotoxin-insensitive vesicular-associated membrane protein (TI-VAMP/VAMP7), a vesicular SNARE expressed in several cell types including neurons, was previously shown to play a major role in exocytosis involved in neurite growth in cultured neurons. Here we generated a complete constitutive knock-out by deleting the exon 3 ofVamp7. Loss of TI-VAMP expression did not lead to any striking developmental or neurological defect. Knock-out mice displayed decreased brain weight and increased third ventricle volume. Axon growth appeared normal in cultured knock-out neurons. Behavioral characterization unraveled that TI-VAMP knock-out was associated with increased anxiety. Our results thus suggest compensatory mechanisms allowing the TI-VAMP knock-out mice to fulfill major developmental processes. The phenotypic traits unraveled here further indicate an unexpected role of TI-VAMP-mediated vesicular traffic in anxiety and suggest a role for TI-VAMP in higher brain functions.

Integrative and conjugative elements (ICEs), also called conjugative transposons, are genomic islands that excise, self-transfer by conjugation, and integrate in the genome of the recipient bacterium. The current investigation shows the intraspecies conjugative transfer of the first described ICEs in Streptococcus thermophilus , ICE St1 and ICE St3 . Mitomycin C, a DNA-damaging agent, derepresses ICE St3 conjugative transfer almost 25-fold. The ICE St3 host range was determined using various members of the Firmicutes as recipients. Whereas numerous ICE St3 transconjugants of Streptococcus pyogenes and Enterococcus faecalis were recovered, only one transconjugant of Lactococcus lactis was obtained. The newly incoming ICEs, except the one from L. lactis , are site-specifically integrated into the 3′ end of the fda gene and are still able to excise in these transconjugants. Furthermore, ICE St3 was retransferred from E. faecalis to S. thermophilus . Recombinant plasmids carrying different parts of the ICE St1 recombination module were used to show that the integrase gene is required for the site-specific integration and excision of the ICEs, whereas the excisionase gene is required for the site-specific excision only.

In this work, four types of genomic islands related to ICESt1 were characterized. They are integrated in the same location as ICESt1 in seven other strains of S. thermophilus. One of these elements, ICESt3, is probably an ICE. Two other types of elements, CIME19258 and CIME302, are flanked by site-specific attachment sites closely related to attL and attR of ICESt1 and ICESt3 whereas DCIME308 only possesses a putative attR site. They have arisen from ICEs by deletion of there conjugation and recombination modules. Comparisons between ICE and CIME (cis-mobilizable element) from Streptococcus thermophilus and functional analysis, using plasmids harbouring the entire or a part of the recombination module of ICESt1, suggest that these elements evolve by a novel model.). The circular form of an ICE, acquired by conjugation, integrates by site-specific in the attR site of the CIME, leading to the structure attL-CIME-attI'-ICE-attR'. Then partial or entire deletion of the attI site would stabilise the composite element. The whole structure would be a novel ICE and could be excised by recombination between attL and attR and transferred to a new host bacterium. This suggests that ICEs have acquired novel modules by a new mechanism : accretion / mobilization of CIMEs. In conclusion, ICE and probably CIME would be important in the evolution of bacterial genomes by horizontal gene transfer and contribute to the acquisition of new functions by these genomes., Lors de ce travail, quatre types d'îlots génomiques apparentés à ICESt1 ont été caractérisés. Ils sont intégrés au même site que lui chez sept souches de S. thermophilus. L'un des éléments, ICESt3 est probablement un ICE. Deux autres types d'éléments, CIME19258 et CIME302 sont flanqués par des sites attL et attR apparentés à ceux d'ICESt1 et d'ICESt3 tandis que DCIME308 possède uniquement un site attR. Ces éléments dérivent d'un ICE par délétion des modules de conjugaison et de recombinaison. La comparaison des ICE et CIME (CIs Mobilisable Element ) de S. thermophilus et des études fonctionnelles réalisées avec des plasmides portant tout ou partie du module de recombinaison d'ICESt1 suggèrent que ces éléments évoluent selon un modèle original. La forme circulaire d'un ICE acquise par conjugaison s'intégrerait par recombinaison site-spécifique dans le site attR du CIME formant ainsi une structure attL-CIME-attI-ICE-attR. Le site attI serait ensuite partiellement ou totalement délété stabilisant la structure. Cet ensemble constituerait un nouvel ICE qui s'exciserait par recombinaison site-spécifique puis se transférerait par conjugaison. Les ICE acquerraient ainsi de nouveaux modules par un mécanisme inconnu jusqu'alors : l'accrétion et la mobilisation site-spécifique de CIME. En conclusion, les ICE et probablement les CIME joueraient un rôle important dans l'évolution par transfert horizontal des génomes bactériens et participeraient à l'acquisition de nouvelles fonctions par ces génomes.

International audience; The 34,734-bp element ICESt1 from Streptococcus thermophilus CNRZ368 is site-specifically integrated into the 3(') end of the gene fda. ICESt1 encodes integrative functions and putative transfer functions. Six proteins of the putative conjugative system of ICESt1 are related to those encoded by the conjugative transposon Tn916 from Enterococcus faecalis. A comparison of these proteins with those encoded by the complete or partial genome sequences of various low G+C bacteria including Bacillus subtilis, Clostridium difficile, E. faecalis, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus mutans revealed the presence of numerous putative site-specific integrative conjugative elements and/or conjugative transposons within these genomes. Sequence comparisons revealed that these elements possess a modular structure and that exchanges of unrelated or distantly related modules and genes have occurred between these elements, and also plasmids and prophages. These exchanges have probably led to modifications in the site specificity of integration of these elements. Therefore, a distinction between low specificity integrative conjugative elements (i.e., conjugative transposons) and site-specific integrative conjugative elements does not appear to be relevant. We propose to call all the conjugative elements that excise by site-specific recombination and integrate by recombination between a specific site of a circular intermediate and another site, "Integrative and Conjugative Elements" (ICEs), irrespective of the integration specificity.

International audience; A 34.7-kb element, ICESt1, is integrated in the 3' end of fda locus from Streptococcus thermophilus CNRZ368. ICESt1 excises by a site-specific recombination between two 27-pb identical sequences flanking the element. It encodes an integrase required for excision. Furthermore, eleven putative proteins encoded by ICESt1 are related to proteins encoded by various conjugative elements from low G + C Gram positive bacteria. Therefore, ICESt1 could be a site-specific integrative conjugative element (ICE). Comparison of proteins encoded by ICESt1 and the sequenced genome of Bacillus subtilis 168 revealed a putative 20.5-kb ICE, ICEBs1. Sequence comparison of ICESt1, ICEBs1, Tn916 and Tn5252 revealed exchanges of modules between ICEs, conjugative transposons and prophages. Four types of elements related to ICESt1 (IEs) were found in seven other strains of S. thermophilus and are integrated in the same location as ICESt1. One of these elements, IE385, could be an ICE whereas the others do not seem to be integrative and conjugative. Comparison of the various elements and ICESt1 showed that all of them have a chimerical structure resulting from exchanges of regions from different origins. The left end of IE19258 is identical to an internal recombination site of ICESt1, attL', but shares only 57% identity with its left end, attL. The site-specific recombination between the cores of attL' and of the right end, attR, leads to the excision of a circular molecule corresponding to the region flanked by these sites. Therefore, this suggests that ICESt1 results from the integration of a 28.2-kb ICE, ICESt2, in the attR' site of an IE element and that ICESt2 have mobilized the IE.; Caractérisation et structure chimérique d'une famille d'éléments intégratifs potentiellement conjugatifs chez Streptococcus thermophilus . Un élément de 34,7 kb, ICESt1, est intégré dans l'extrémité 3' du locus fda de Streptococcus thermophilus CNRZ368. ICESt1 s'excise par recombinaison site-spécifique entre des séquences identiques de 27 pb flanquant l'élément. ICESt1 code une intégrase nécessaire à cette excision. Onze des ORF d'ICESt1 codent des protéines apparentées à celles codées par divers éléments conjugatifs de bactéries Gram positives à bas G + C. ICESt1 serait donc un élément conjugatif à intégration site-spécifique (ICE). La comparaison des protéines codées par ICESt1 et le génome séquencé de Bacillus subtilis 168 a révélé un ICE de 20,5 kb, ICEBs1. L'analyse des séquences d'ICESt1, ICEBs1, Tn916 et Tn5252 révèle des échanges de modules entre éléments conjugatifs intégratifs, transposons conjugatifs et prophages. Par ailleurs, 4 types d'éléments apparentés à ICESt1 d'une taille de 12,8 à 26,1 kb (IE) sont intégrés exactement au même site qu'ICESt1 chez 7 autres souches de S. thermophilus. Seul, IE385 serait un ICE. Les autres éléments ne semblent ni conjugatifs ni intégratifs. Les hybridations entre éléments et le séquençage partiel montrent que chacun des éléments possède une structure chimérique complexe associant des régions d'origines différentes. L'extrémité gauche d'IE19258 est identique à un site de recombinaison interne d'ICESt1, attL', mais ne présente que 57 % d'identité avec son extrémité gauche. La recombinaison site spécifique entre attL' et l'extrémité droite attR d'ICESt1 conduit à l'excision d'une forme circulaire de la région comprise entre les 2 sites. Ceci indique qu'ICESt1 serait constitué de 2 éléments : un IE compris entre attL et attL' et un élément conjugatif intégratif de 28,2 kb compris entre attL' et attR, ICESt2, qui se serait intégré à la frontière de l'IE et l'aurait mobilisé.

Two large-scale phenotyping efforts, the European Mouse Disease Clinic (EUMODIC) and the Wellcome Trust Sanger Institute Mouse Genetics Project (SANGER-MGP), started during the late 2000s with the aim to deliver a comprehensive assessment of phenotypes or to screen for robust indicators of diseases in mouse mutants. They both took advantage of available mouse mutant lines but predominantly of the embryonic stem (ES) cells resources derived from the European Conditional Mouse Mutagenesis programme (EUCOMM) and the Knockout Mouse Project (KOMP) to produce and study 799 mouse models that were systematically analysed with a comprehensive set of physiological and behavioural paradigms. They captured more than 400 variables and an additional panel of metadata describing the conditions of the tests. All the data are now available through EuroPhenome database ( www.europhenome.org ) and the WTSI mouse portal ( http://www.sanger.ac.uk/mouseportal/ ), and the corresponding mouse lines are available through the European Mouse Mutant Archive (EMMA), the International Knockout Mouse Consortium (IKMC), or the Knockout Mouse Project (KOMP) Repository. Overall conclusions from both studies converged, with at least one phenotype scored in at least 80% of the mutant lines. In addition, 57% of the lines were viable, 13% subviable, 30% embryonic lethal, and 7% displayed fertility impairments. These efforts provide an important underpinning for a future global programme that will undertake the complete functional annotation of the mammalian genome in the mouse model.

The 34 734-bp integrative and potentially conjugative element (putative ICE) ICESt1 has been previously found to be site-specifically integrated in the 3′ end of the fda locus of Streptococcus thermophilus CNRZ368. Four types of genomic islands related to ICESt1 are integrated in the same position in seven other strains of S. thermophilus. One of these elements, ICESt3, harbours conjugation and recombination modules closely related to those of ICESt1 and excises by site-specific recombination. Two other types of elements, CIME19258 and CIME302, are flanked by site-specific attachment sites closely related to attL and attR of ICESt1 and ICESt3, whereas ΔCIME308 only possesses a putative attR site; none of these three elements carry complete conjugation and recombination modules. ICESt1 contains a functional internal recombination site, attL′, that is almost identical to attL of CIME19258. The recombination between attL′ and attR of ICESt1 leads to the excision of the expected circular molecule (putative ICE); a cis-mobilizable element (CIME) flanked by an attL site and an attB′ site remains integrated into the 3′ end of fda. Furthermore, sequences that could be truncated att sites were found within ICESt1, ICESt3 and CIME302. All together, these data suggest that these genomic islands evolved by deletion and tandem accretion of ICEs and CIMEs resulting from site-specific recombination. A model for this evolution is proposed and its application to other genomic islands is discussed.

The identification of causal variants in sequencing studies remains a considerable challenge that can be partially addressed by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and essentiality screens carried out on human cell lines. We propose a cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing biological properties. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented among genes non-essential for cell survival but required for organism development. After screening developmental disorder cases from three independent disease sequencing consortia, we identify potentially pathogenic variants in genes not previously associated with rare diseases. We therefore propose FUSIL as an efficient approach for disease gene discovery., Discovery of causal variants for monogenic disorders has been facilitated by whole exome and genome sequencing, but does not provide a diagnosis for all patients. Here, the authors propose a Full Spectrum of Intolerance to Loss-of-Function (FUSIL) categorization that integrates gene essentiality information to aid disease gene discovery.

The role of sex in biomedical studies has often been overlooked, despite evidence of sexually dimorphic effects in some biological studies. Here, we used high-throughput phenotype data from 14,250 wildtype and 40,192 mutant mice (representing 2,186 knockout lines), analysed for up to 234 traits, and found a large proportion of mammalian traits both in wildtype and mutants are influenced by sex. This result has implications for interpreting disease phenotypes in animal models and humans., Systemic dissection of sexually dimorphic phenotypes in mice is lacking. Here, Karp and the International Mouse Phenotype Consortium show that approximately 10% of qualitative traits and 56% of quantitative traits in mice as measured in laboratory setting are sexually dimorphic.