The circadian gene Arntl2 on distal mouse chromosome 6 controls thymocyte apoptosis.

Nonobese diabetic (NOD) mice are a model for type 1 diabetes that displays defects in central immune tolerance, including impairment of thymocyte apoptosis and proliferation. Thymocyte apoptosis is decreased in NOD/Lt mice compared to nondiabetic C3H/HeJ and C57BL/6 mice. Analysis of a set of NOD.C3H and NOD.B6 congenic mouse strains for distal chromosome 6 localizes the phenotype to the 700 kb Idd6.3 interval. Idd6.3 contains the type 1 diabetes candidate gene aryl hydrocarbon receptor nuclear translocator-like 2 (Arntl2), encoding a circadian rhythm-related transcription factor. Newly generated Arntl2 -/- mouse strains reveal that inactivation of the B6 allele of Arntl2 is sufficient to both decrease thymocyte apoptosis and proliferation. When expressed from C3H or B6 alleles, ARNTL2 inhibits the transcription of interleukin 21 (Il21), a major player in the regulation of immune responses. IL-21 injection abolishes the B6 allele-mediated decrease of apoptosis and proliferation. Interestingly, IL-21 also leads to an increase in thymic proinflammatory Th17 helper cells. Our results identify Arntl2 as a gene controlling thymocyte apoptosis and proliferation along with Th17 development through the IL-21 pathway.

A novel nodal enhancer dependent on pluripotency factors and smad2/3 signaling conditions a regulatory switch during epiblast maturation.

During early development, modulations in the expression of Nodal, a TGFß family member, determine the specification of embryonic and extra-embryonic cell identities. Nodal has been extensively studied in the mouse, but aspects of its early expression remain unaccounted for. We identified a conserved hotspot for the binding of pluripotency factors at the Nodal locus and called this sequence "highly bound element" (HBE). Luciferase-based assays, the analysis of fluorescent HBE reporter transgenes, and a conditional mutation of HBE allowed us to establish that HBE behaves as an enhancer, is activated ahead of other Nodal enhancers in the epiblast, and is essential to Nodal expression in embryonic stem cells (ESCs) and in the mouse embryo. We also showed that HBE enhancer activity is critically dependent on its interaction with the pluripotency factor Oct4 and on Activin/Nodal signaling. Use of an in vitro model of epiblast maturation, relying on the differentiation of ESCs into epiblast stem cells (EpiSCs), revealed that this process entails a shift in the regulation of Nodal expression from an HBE-driven phase to an ASE-driven phase, ASE being another autoregulatory Nodal enhancer. Deletion of HBE in ESCs or in EpiSCs allowed us to show that HBE, although not necessary for Nodal expression in EpiSCs, is required in differentiating ESCs to activate the differentiation-promoting ASE and therefore controls this regulatory shift. Our findings clarify how early Nodal expression is regulated and suggest how this regulation can promote the specification of extra-embryonic precusors without inducing premature differentiation of epiblast cells. More generally, they open new perspectives on how pluripotency factors achieve their function.

Human hematopoietic reconstitution and HLA-restricted responses in nonpermissive alymphoid mice.

We generated a new humanized mouse model to study HLA-restricted immune responses. For this purpose, we created unique murine hosts by enforcing the expression of human SIRPα by murine phagocytes in murine MHC-deficient HLA-transgenic alymphoid hosts, an approach that allowed the immune reconstitution of nonpermissive mice following injection of human hematopoietic stem cells. We showed that these mouse/human chimeras were able to generate HLA-restricted responses to immunization. These new humanized mice may offer attractive models to study immune responses to human diseases, such as HIV and EBV infections, as well as to assay new vaccine strategies.

Mutations in the gene encoding pejvakin, a newly identified protein of the afferent auditory pathway, cause DFNB59 auditory neuropathy.

Auditory neuropathy is a particular type of hearing impairment in which neural transmission of the auditory signal is impaired, while cochlear outer hair cells remain functional. Here we report on DFNB59, a newly identified gene on chromosome 2q31.1-q31.3 mutated in four families segregating autosomal recessive auditory neuropathy. DFNB59 encodes pejvakin, a 352-residue protein. Pejvakin is a paralog of DFNA5, a protein of unknown function also involved in deafness. By immunohistofluorescence, pejvakin is detected in the cell bodies of neurons of the afferent auditory pathway. Furthermore, Dfnb59 knock-in mice, homozygous for the R183W variant identified in one DFNB59 family, show abnormal auditory brainstem responses indicative of neuronal dysfunction along the auditory pathway. Unlike previously described sensorineural deafness genes, all of which underlie cochlear cell pathologies, DFNB59 is the first human gene implicated in nonsyndromic deafness due to a neuronal defect.

Protective role of LGP2 in influenza virus pathogenesis.

Influenza A virus triggers a contagious respiratory disease that can cause considerable morbidity and mortality. Using an in vitro approach, we previously demonstrated that the pattern recognition receptor retinoic acid-inducible gene I (RIG-I) plays a key role in influenza A virus-mediated immune response. However, the importance of RIG-I signaling in vivo has not been thoroughly examined, because of the lack of an appropriate mouse models. To circumvent this issue, we generated a new transgenic mouse overexpressing LGP2 (hereafter, "LGP2 TG mice"), a major regulator of the RIG-I signaling pathway. The time course of several parameters was compared in infected wild-type and LGP2 TG mice. We found that LGP2 TG mice displayed significantly reduced inflammatory mediators and a lower leukocyte infiltration into the bronchoalveolar airspace. More importantly, LGP2 TG mice had a significant survival advantage. Hence, our in vivo study reveals that LGP2 is a major downregulator of the influenza A virus-triggered detrimental inflammatory response.

Mouse PRDM9 DNA-binding specificity determines sites of histone H3 lysine 4 trimethylation for initiation of meiotic recombination.

Meiotic recombination generates reciprocal exchanges between homologous chromosomes (also called crossovers, COs) that are essential for proper chromosome segregation during meiosis and are a major source of genome diversity by generating new allele combinations. COs have two striking properties: they occur at specific sites, called hotspots, and these sites evolve rapidly. In mammals, the Prdm9 gene, which encodes a meiosis-specific histone H3 methyltransferase, has recently been identified as a determinant of CO hotspots. Here, using transgenic mice, we show that the sole modification of PRDM9 zinc fingers leads to changes in hotspot activity, histone H3 lysine 4 trimethylation (H3K4me3) levels, and chromosome-wide distribution of COs. We further demonstrate by an in vitro assay that the PRDM9 variant associated with hotspot activity binds specifically to DNA sequences located at the center of the three hotspots tested. Remarkably, we show that mutations in cis located at hotspot centers and associated with a decrease of hotspot activity affect PRDM9 binding. Taken together, these results provide the direct demonstration that Prdm9 is a master regulator of hotspot localization through the DNA binding specificity of its zinc finger array and that binding of PRDM9 at hotspots promotes local H3K4me3 enrichment.

Progressive Purkinje cell degeneration in tambaleante mutant mice is a consequence of a missense mutation in HERC1 E3 ubiquitin ligase.

The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domains have been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a G<-->A transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N-terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology.

A fast Myosin super enhancer dictates muscle fiber phenotype through competitive interactions with Myosin genes.

The contractile properties of adult myofibers are shaped by their Myosin heavy chain isoform content. Here, we identify by snATAC-seq a 42 kb super-enhancer at the locus regrouping the fast Myosin genes. By 4C-seq we show that active fast Myosin promoters interact with this super-enhancer by DNA looping, leading to the activation of a single promoter per nucleus. A rainbow mouse transgenic model of the locus including the super-enhancer recapitulates the endogenous spatio-temporal expression of adult fast Myosin genes. In situ deletion of the super-enhancer by CRISPR/Cas9 editing demonstrates its major role in the control of associated fast Myosin genes, and deletion of two fast Myosin genes at the locus reveals an active competition of the promoters for the shared super-enhancer. Last, by disrupting the organization of fast Myosin, we uncover positional heterogeneity within limb skeletal muscles that may underlie selective muscle susceptibility to damage in certain myopathies.

Dynamics of myogenic differentiation using a novel Myogenin knock-in reporter mouse.

BACKGROUND: Myogenin is a transcription factor that is expressed during terminal myoblast differentiation in embryonic development and adult muscle regeneration. Investigation of this cell state transition has been hampered by the lack of a sensitive reporter to dynamically track cells during differentiation. RESULTS: Here, we report a knock-in mouse line expressing the tdTOMATO fluorescent protein from the endogenous Myogenin locus. Expression of tdTOMATO in MyogntdTom mice recapitulated endogenous Myogenin expression during embryonic muscle formation and adult regeneration and enabled the isolation of the MYOGENIN+ cell population. We also show that tdTOMATO fluorescence allows tracking of differentiating myoblasts in vitro and by intravital imaging in vivo. Lastly, we monitored by live imaging the cell division dynamics of differentiating myoblasts in vitro and showed that a fraction of the MYOGENIN+ population can undergo one round of cell division, albeit at a much lower frequency than MYOGENIN- myoblasts. CONCLUSIONS: We expect that this reporter mouse will be a valuable resource for researchers investigating skeletal muscle biology in developmental and adult contexts.

The antitumoral effects of chemerin are independent from leukocyte recruitment and mediated by inhibition of neoangiogenesis.

Chemerin, a multifunctional protein acting through the receptor ChemR23/CMKLR1, is downregulated in various human tumors and was shown to display antitumoral properties in mouse models of cancer. In the present study, we report that bioactive chemerin expression by tumor cells delays the growth of B16 melanoma and Lewis lung carcinoma in vivo. A similar delay is observed when chemerin is not expressed by tumor cells but by keratinocytes of the host mice. The protective effect of chemerin is mediated by CMKLR1 and appears unrelated to the recruitment of leukocyte populations. Rather, tumors grown in the presence of chemerin display a much smaller number of blood vessels, hypoxic regions early in their development, and larger necrotic areas. These observations likely explain the slower growth of the tumors. The anti-angiogenic effects of chemerin were confirmed in a bead sprouting assay using human umbilical vein endothelial cells. These results suggest that CMKLR1 agonists might constitute therapeutic molecules inhibiting the neoangiogenesis process in solid tumors.

The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium.

The R2TP chaperone cooperates with HSP90 to integrate newly synthesized proteins into multi-subunit complexes, yet its role in tissue homeostasis is unknown. Here, we generated conditional, inducible knock-out mice for Rpap3 to inactivate this core component of R2TP in the intestinal epithelium. In adult mice, Rpap3 invalidation caused destruction of the small intestinal epithelium and death within 10 days. Levels of R2TP substrates decreased, with strong effects on mTOR, ATM and ATR. Proliferative stem cells and progenitors deficient for Rpap3 failed to import RNA polymerase II into the nucleus and they induced p53, cell cycle arrest and apoptosis. Post-mitotic, differentiated cells did not display these alterations, suggesting that R2TP clients are preferentially built in actively proliferating cells. In addition, high RPAP3 levels in colorectal tumors from patients correlate with bad prognosis. Here, we show that, in the intestine, the R2TP chaperone plays essential roles in normal and tumoral proliferation.

Corrigendum: Expression of Bioactive Chemerin by Keratinocytes Inhibits Late Stages of Tumor Development in a Chemical Model of Skin Carcinogenesis.

[This corrects the article DOI: 10.3389/fonc.2019.01253.].

A distinct cardiopharyngeal mesoderm genetic hierarchy establishes antero-posterior patterning of esophagus striated muscle.

In most vertebrates, the upper digestive tract is composed of muscularized jaws linked to the esophagus that permits food ingestion and swallowing. Masticatory and esophagus striated muscles (ESM) share a common cardiopharyngeal mesoderm (CPM) origin, however ESM are unusual among striated muscles as they are established in the absence of a primary skeletal muscle scaffold. Using mouse chimeras, we show that the transcription factors Tbx1 and Isl1 are required cell-autonomously for myogenic specification of ESM progenitors. Further, genetic loss-of-function and pharmacological studies point to MET/HGF signaling for antero-posterior migration of esophagus muscle progenitors, where Hgf ligand is expressed in adjacent smooth muscle cells. These observations highlight the functional relevance of a smooth and striated muscle progenitor dialogue for ESM patterning. Our findings establish a Tbx1-Isl1-Met genetic hierarchy that uniquely regulates esophagus myogenesis and identify distinct genetic signatures that can be used as framework to interpret pathologies arising within CPM derivatives.

Expression of Bioactive Chemerin by Keratinocytes Inhibits Late Stages of Tumor Development in a Chemical Model of Skin Carcinogenesis.

Chemerin is a multifunctional protein acting mainly through the G protein-coupled receptor ChemR23/CMKLR1/Chemerin1. Its expression is frequently downregulated in human tumors, including in melanoma and squamous cell carcinoma of the skin and anti-tumoral properties of chemerin were reported in mouse tumor graft models. In the present study, we report the development of spontaneous skin tumors in aged ChemR23-deficient mice. In order to test the potential therapeutic benefit of chemerin analogs, a transgenic model in which bioactive chemerin is over-expressed by basal keratinocytes was generated. These animals are characterized by increased levels of chemerin immunoreactivity and bioactivity in the skin and the circulation. In a chemical carcinogenesis model, papillomas developed later, were less numerous, and their progression to carcinomas was delayed. Temporal control of chemerin expression by doxycycline allowed to attribute its effects to late stages of carcinogenesis. The protective effects of chemerin were partly abrogated by ChemR23 invalidation. These results demonstrate that chemerin is able to delay very significantly tumor progression in a model that recapitulates closely the evolution of solid cancer types in human and suggest that the chemerin-ChemR23 system might constitute an interesting target for therapeutic intervention in the cancer field.

Generation of an Msx2-GFP conditional null allele.

Msx1 and Msx2, two members of the Msx gene family, encode homeoprotein transcription factors and play critical roles during mouse development. Because of the redundancy between the two genes, many of these roles can only be studied in double Msx1; Msx2 mutants. However, these animals die around 14.5 dpc, which precludes analysis of Msx gene function beyond this stage. Moreover, the pleiotropic defects displayed by these embryos make phenotypic analysis difficult. To overcome these restrictions and study the double Msx mutant phenotype at later stages, we generated an Msx2 conditional null allele using Cre/loxP technology. The strategy consisted of flanking the Msx2 gene coding sequence with two loxP sites. In addition, a green fluorescent protein (GFP) reporter gene was placed under Msx2 regulatory sequences in the modified locus. Our results demonstrate that the Msx2-GFP conditional allele behaves as a normal one, whereas Cre-mediated recombination creates an Msx2 null allele. With either allele, expression patterns of the GFP reporter gene and the Msx2 endogenous gene are identical.

A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats.

Tobacco addiction is a chronic and relapsing disorder with an important genetic component that represents a major public health issue. Meta-analysis of large-scale human genome-wide association studies (GWASs) identified a frequent non-synonymous SNP in the gene coding for the α5 subunit of nicotinic acetylcholine receptors (α5SNP), which significantly increases the risk for tobacco dependence and delays smoking cessation. To dissect the neuronal mechanisms underlying the vulnerability to nicotine addiction in carriers of the α5SNP, we created rats expressing this polymorphism using zinc finger nuclease technology and evaluated their behavior under the intravenous nicotine-self-administration paradigm. The electrophysiological responses of their neurons to nicotine were also evaluated. α5SNP rats self-administered more nicotine at high doses and exhibited higher nicotine-induced reinstatement of nicotine seeking than wild-type rats. Higher reinstatement was associated with altered neuronal activity in several discrete areas that are interconnected, including in the interpeduncular nucleus (IPN), a GABAergic structure that strongly expresses α5-containing nicotinic receptors. The altered reactivity of IPN neurons of α5SNP rats to nicotine was confirmed electrophysiologically. In conclusion, the α5SNP polymorphism is a major risk factor for nicotine intake at high doses and for relapse to nicotine seeking in rats, a dual effect that reflects the human condition. Our results also suggest an important role for the IPN in the higher relapse to nicotine seeking observed in α5SNP rats.

Generation of a Double KO Mouse by Simultaneous Targeting of the Neighboring Genes Tmem176a and Tmem176b Using CRISPR/Cas9: Key Steps from Design to Genotyping.

The CRISPR/Cas9 system has been tailored to a revolutionary genetic tool because of its remarkable simplicity and efficacy. While complex genome editing in the mouse since the 1990s has been dominated by the use of embryonic stem (ES) cells, CRISPR/Cas9 now offers a versatile and fast approach to precisely modify virtually any DNA regions directly in mouse zygotes. Yet, this relative simplicity does not preclude a conscientious preparatory work that is often neglected when initiating a project. Here, we describe the key steps leading to successful generation of a double knockout (KO) mouse by simultaneously targeting two homolog genes, Tmem176a and Tmem176b, which are located in the same genomic locus. Additionally, we show that similar efficiency can be obtained in a mixed genetic background or directly in the C57BL/6 inbred strain. Thus, presented as a detailed case study that should be helpful to the non-specialists, we focus on the genotyping strategy to anticipate the various possibilities.

Implementation of meiosis prophase I programme requires a conserved retinoid-independent stabilizer of meiotic transcripts.

Sexual reproduction is crucially dependent on meiosis, a conserved, specialized cell division programme that is essential for the production of haploid gametes. Here we demonstrate that fertility and the implementation of the meiotic programme require a previously uncharacterized meiosis-specific protein, MEIOC. Meioc invalidation in mice induces early and pleiotropic meiotic defects in males and females. MEIOC prevents meiotic transcript degradation and interacts with an RNA helicase that binds numerous meiotic mRNAs. Our results indicate that proper engagement into meiosis necessitates the specific stabilization of meiotic transcripts, a previously little-appreciated feature in mammals. Remarkably, the upregulation of MEIOC at the onset of meiosis does not require retinoic acid and STRA8 signalling. Thus, we propose that the complete induction of the meiotic programme requires both retinoic acid-dependent and -independent mechanisms. The latter process involving post-transcriptional regulation likely represents an ancestral mechanism, given that MEIOC homologues are conserved throughout multicellular animals.

The HERC2 ubiquitin ligase is essential for embryonic development and regulates motor coordination.

A mutation in the HERC2 gene has been linked to a severe neurodevelopmental disorder with similarities to the Angelman syndrome. This gene codifies a protein with ubiquitin ligase activity that regulates the activity of tumor protein p53 and is involved in important cellular processes such as DNA repair, cell cycle, cancer, and iron metabolism. Despite the critical role of HERC2 in these physiological and pathological processes, little is known about its relevance in vivo. Here, we described a mouse with targeted inactivation of the Herc2 gene. Homozygous mice were not viable. Distinct from other ubiquitin ligases that interact with p53, such as MDM2 or MDM4, p53 depletion did not rescue the lethality of homozygous mice. The HERC2 protein levels were reduced by approximately one-half in heterozygous mice. Consequently, HERC2 activities, including ubiquitin ligase and stimulation of p53 activity, were lower in heterozygous mice. A decrease in HERC2 activities was also observed in human skin fibroblasts from individuals with an Angelman-like syndrome that express an unstable mutant protein of HERC2. Behavioural analysis of heterozygous mice identified an impaired motor synchronization with normal neuromuscular function. This effect was not observed in p53 knockout mice, indicating that a mechanism independent of p53 activity is involved. Morphological analysis showed the presence of HERC2 in Purkinje cells and a specific loss of these neurons in the cerebella of heterozygous mice. In these animals, an increase of autophagosomes and lysosomes was observed. Our findings establish a crucial role of HERC2 in embryonic development and motor coordination.