RESUMEN
Coenzyme Q (CoQ) deficiency syndrome is conventionally treated with limited efficacy using exogenous CoQ10. Poor outcomes result from low absorption and bioavailability of CoQ10 and the clinical heterogenicity of the disease. Here, we demonstrate that supplementation with 4-hydroxybenzoic acid (4HB), the precursor of the benzoquinone ring in the CoQ biosynthetic pathway, completely rescues multisystemic disease and perinatal lethality in a mouse model of CoQ deficiency. 4HB stimulates endogenous CoQ biosynthesis in tissues of Coq2 mutant mice, normalizing mitochondrial function and rescuing cardiac insufficiency, edema, and neurodevelopmental delay. In contrast, exogenous CoQ10 supplementation falls short in fully restoring the phenotype. The treatment is translatable to human use, as proven by in vitro studies in skin fibroblasts from patients with pathogenic variants in COQ2. The therapeutic approach extends to other disorders characterized by deficiencies in the production of 4HB and early steps of CoQ biosynthesis and instances of secondary CoQ deficiency.
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Modelos Animales de Enfermedad , Enfermedades Mitocondriales , Parabenos , Ubiquinona , Animales , Enfermedades Mitocondriales/tratamiento farmacológico , Enfermedades Mitocondriales/patología , Enfermedades Mitocondriales/metabolismo , Parabenos/farmacología , Ubiquinona/análogos & derivados , Ubiquinona/farmacología , Ubiquinona/metabolismo , Ubiquinona/deficiencia , Ratones , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Humanos , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Ratones Endogámicos C57BL , Debilidad Muscular/tratamiento farmacológico , Debilidad Muscular/metabolismo , Debilidad Muscular/patología , Ataxia/tratamiento farmacológico , Ataxia/patología , Ataxia/metabolismoRESUMEN
It has been established for almost 30 years that the retinoic acid receptor (RAR) signalling pathway plays essential roles in the morphogenesis of a large variety of organs and systems. Here, we used a temporally controlled genetic ablation procedure to precisely determine the time windows requiring RAR functions. Our results indicate that from E8.5 to E9.5, RAR functions are critical for the axial rotation of the embryo, the appearance of the sinus venosus, the modelling of blood vessels, and the formation of forelimb buds, lung buds, dorsal pancreatic bud, lens, and otocyst. They also reveal that E9.5 to E10.5 spans a critical developmental period during which the RARs are required for trachea formation, lung branching morphogenesis, patterning of great arteries derived from aortic arches, closure of the optic fissure, and growth of inner ear structures and of facial processes. Comparing the phenotypes of mutants lacking the 3 RARs with that of mutants deprived of all-trans retinoic acid (ATRA) synthesising enzymes establishes that cardiac looping is the earliest known morphogenetic event requiring a functional ATRA-activated RAR signalling pathway.
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Major progress has been made over the last decade in identifying novel genes involved in neurodevelopmental disorders, although the task of elucidating their corresponding molecular and pathophysiological mechanisms, which are an essential prerequisite for developing therapies, has fallen far behind. We selected 45 genes for intellectual disabilities to generate and characterize mouse models. Thirty-nine of them were based on the frequency of pathogenic variants in patients and literature reports, with several corresponding to de novo variants, and six other candidate genes. We used an extensive screen covering the development and adult stages, focusing specifically on behaviour and cognition to assess a wide range of functions and their pathologies, ranging from basic neurological reflexes to cognitive abilities. A heatmap of behaviour phenotypes was established, together with the results of selected mutants. Overall, three main classes of mutant lines were identified based on activity phenotypes, with which other motor or cognitive deficits were associated. These data showed the heterogeneity of phenotypes between mutation types, recapitulating several human features, and emphasizing the importance of such systematic approaches for both deciphering genetic etiological causes of ID and autism spectrum disorders, and for building appropriate therapeutic strategies.
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3D imaging in animal models, during development or in adults, facilitates the identification of structural morphological changes that cannot be achieved with traditional 2D histological staining. Through the reconstruction of whole embryos or a region-of-interest, specific changes are better delimited and can be easily quantified. We focused here on high-resolution episcopic microscopy (HREM), and its potential for visualizing and quantifying the organ systems of normal and genetically altered embryos and adult organisms. Although the technique is based on episcopic images, these are of high resolution and are close to histological quality. The images reflect the tissue structure and densities revealed by histology, albeit in a grayscale color map. HREM technology permits researchers to take advantage of serial 2D aligned stacks of images to perform 3D reconstructions. Three-dimensional visualization allows for an appreciation of topology and morphology that is difficult to achieve with classical histological studies. The nature of the data lends itself to novel forms of computational analysis that permit the accurate quantitation and comparison of individual embryos in a manner that is impossible with histology. Here, we have developed a new HREM prototype consisting of the assembly of a Leica Biosystems Nanocut rotary microtome with optics and a camera. We describe some examples of applications in the prenatal and adult lifestage of the mouse to show the added value of HREM for phenotyping experimental cohorts to compare and quantify structure volumes. At prenatal stages, segmentations and 3D reconstructions allowed the quantification of neural tissue and ventricular system volumes of normal brains at E14.5 and E16.5 stages. 3D representations of normal cranial and peripheric nerves at E15.5 and of the normal urogenital system from stages E11.5 to E14.5 were also performed. We also present a methodology to quantify the volume of the atherosclerotic plaques of ApoEtm1Unc/tm1Unc mutant mice and illustrate a 3D reconstruction of knee ligaments in adult mice.
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Anorectal malformations (ARMs) are relatively common congenital abnormalities, but their pathogenesis is poorly understood. Previous gene knockout studies indicated that the signalling pathway mediated by the retinoic acid receptors (RAR) is instrumental to the formation of the anorectal canal and of various urogenital structures. Here, we show that simultaneous ablation of the three RARs in the mouse embryo results in a spectrum of malformations of the pelvic organs in which anorectal and urinary bladder ageneses are consistently associated. We found that these ageneses could be accounted for by defects in the processes of growth and migration of the cloaca, the embryonic structure from which the anorectal canal and urinary bladder originate. We further show that these defects are preceded by a failure of the lateral shift of the umbilical arteries and propose vascular abnormalities as a possible cause of ARM. Through the comparisons of these phenotypes with those of other mutant mice and of human patients, we would like to suggest that morphological data may provide a solid base to test molecular as well as clinical hypotheses.
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Skeletal muscle development and regeneration are tightly regulated processes. How the intracellular organization of muscle fibers is achieved during these steps is unclear. Here, we focus on the cellular and physiological roles of amphiphysin 2 (BIN1), a membrane remodeling protein mutated in both congenital and adult centronuclear myopathies (CNM), that is ubiquitously expressed and has skeletal muscle-specific isoforms. We created and characterized constitutive muscle-specific and inducible Bin1 homozygous and heterozygous knockout mice targeting either ubiquitous or muscle-specific isoforms. Constitutive Bin1-deficient mice died at birth from lack of feeding due to a skeletal muscle defect. T-tubules and other organelles were misplaced and altered, supporting a general early role for BIN1 in intracellular organization, in addition to membrane remodeling. Although restricted deletion of Bin1 in unchallenged adult muscles had no impact, the forced switch from the muscle-specific isoforms to the ubiquitous isoforms through deletion of the in-frame muscle-specific exon delayed muscle regeneration. Thus, ubiquitous BIN1 function is necessary for muscle development and function, whereas its muscle-specific isoforms fine tune muscle regeneration in adulthood, supporting that BIN1 CNM with congenital onset are due to developmental defects, whereas later onset may be due to regeneration defects.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Desarrollo de Músculos/fisiología , Músculo Esquelético/fisiología , Proteínas del Tejido Nervioso/metabolismo , Regeneración/fisiología , Proteínas Supresoras de Tumor/metabolismo , Animales , Animales Recién Nacidos , Exones/genética , Conducta Alimentaria , Homocigoto , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/ultraestructura , Especificidad de Órganos , Isoformas de Proteínas/metabolismo , Eliminación de Secuencia , Análisis de SupervivenciaRESUMEN
BAHD1 is a heterochomatinization factor recently described as a component of a multiprotein complex associated with histone deacetylases HDAC1/2. The physiological and patho-physiological functions of BAHD1 are not yet well characterized. Here, we examined the consequences of BAHD1 deficiency in the brains of male mice. While Bahd1 knockout mice had no detectable defects in brain anatomy, RNA sequencing profiling revealed about 2500 deregulated genes in Bahd1-/- brains compared to Bahd1+/+ brains. A majority of these genes were involved in nervous system development and function, behavior, metabolism and immunity. Exploration of the Allen Brain Atlas and Dropviz databases, assessing gene expression in the brain, revealed that expression of the Bahd1 gene was limited to a few territories and cell subtypes, particularly in the hippocampal formation, the isocortex and the olfactory regions. The effect of partial BAHD1 deficiency on behavior was then evaluated on Bahd1 heterozygous male mice, which have no lethal or metabolic phenotypes. Bahd1+/- mice showed anxiety-like behavior and reduced prepulse inhibition (PPI) of the startle response. Altogether, these results suggest that BAHD1 plays a role in chromatin-dependent gene regulation in a subset of brain cells and support recent evidence linking genetic alteration of BAHD1 to psychiatric disorders in a human patient.
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Ansiedad/genética , Encéfalo/metabolismo , Proteínas Cromosómicas no Histona/genética , Reflejo de Sobresalto/genética , Animales , Ansiedad/fisiopatología , Encéfalo/patología , Cromatina/genética , Regulación de la Expresión Génica/genética , Haploinsuficiencia/genética , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , Humanos , Ratones , Ratones Noqueados , Fenotipo , Análisis de Secuencia de ARNRESUMEN
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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Encéfalo/patología , Lisencefalias Clásicas y Heterotopias Subcorticales en Banda/patología , Proteínas Asociadas a Microtúbulos/fisiología , Animales , Lisencefalias Clásicas y Heterotopias Subcorticales en Banda/embriología , Lisencefalias Clásicas y Heterotopias Subcorticales en Banda/genética , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones NoqueadosRESUMEN
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.
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Insuficiencia Multiorgánica/mortalidad , Insuficiencia Multiorgánica/patología , ATPasas de Translocación de Protón/deficiencia , Receptores de Superficie Celular/deficiencia , Animales , Técnicas de Inactivación de Genes , Ratones , Análisis de SupervivenciaRESUMEN
BAHD1 is a vertebrate protein that promotes heterochromatin formation and gene repression in association with several epigenetic regulators. However, its physiological roles remain unknown. Here, we demonstrate that ablation of the Bahd1 gene results in hypocholesterolemia, hypoglycemia and decreased body fat in mice. It also causes placental growth restriction with a drop of trophoblast glycogen cells, a reduction of fetal weight and a high neonatal mortality rate. By intersecting transcriptome data from murine Bahd1 knockout (KO) placentas at stages E16.5 and E18.5 of gestation, Bahd1-KO embryonic fibroblasts, and human cells stably expressing BAHD1, we also show that changes in BAHD1 levels alter expression of steroid/lipid metabolism genes. Biochemical analysis of the BAHD1-associated multiprotein complex identifies MIER proteins as novel partners of BAHD1 and suggests that BAHD1-MIER interaction forms a hub for histone deacetylases and methyltransferases, chromatin readers and transcription factors. We further show that overexpression of BAHD1 leads to an increase of MIER1 enrichment on the inactive X chromosome (Xi). In addition, BAHD1 and MIER1/3 repress expression of the steroid hormone receptor genes ESR1 and PGR, both playing important roles in placental development and energy metabolism. Moreover, modulation of BAHD1 expression in HEK293 cells triggers epigenetic changes at the ESR1 locus. Together, these results identify BAHD1 as a core component of a chromatin-repressive complex regulating placental morphogenesis and body fat storage and suggest that its dysfunction may contribute to several human diseases.
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Proteínas Cromosómicas no Histona/genética , Proteínas Nucleares/genética , Placentación/genética , Esteroides/metabolismo , Factores de Transcripción/genética , Animales , Cromatina/genética , Proteínas Cromosómicas no Histona/biosíntesis , Proteínas de Unión al ADN , Receptor alfa de Estrógeno/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Proteínas Nucleares/biosíntesis , Placenta/metabolismo , Embarazo , Factores de Transcripción/biosíntesis , Transcriptoma/genéticaRESUMEN
OBJECTIVE: Left-ventricular hypertrophy and interstitial fibrosis are the main pathophysiological factors of heart failure with preserved ejection fraction. Blockade of the serotonin 5-HT2B receptor (5-HT2BR) has been shown to reduce cardiac hypertrophy, oxidative stress, and extracellular cell matrix activation. In this study, we evaluated the effects of the 5-HT2BR blockade, on hemodynamic and cardiac remodeling, in spontaneously hypertensive rats (SHRs) that display a diastolic dysfunction with preserved ejection fraction. METHOD: Thirty-seven-week-old SHRs were randomized in four groups receiving either saline, the selective 5-HT2BR antagonist RS-127445 (1âmg/kg per day), a calcium channel blocker nicardipine (6âmg/kg per day), or RS-127445â+ânicardipine. During the 14 weeks of treatment period, cardiac function and blood pressure were monitored by echocardiography and tail-cuff. Finally, electrocardiograms and invasive hemodynamics were obtained before blood collection. Heart was analyzed for morphology and mRNA expression. A complementary study evaluated the cardiac and vascular effects of serotonin on wild-type and mice knockout for the 5-HT2BR (Htr2B) and/or the 5-HT2AR (Htr2A). RESULTS: Despite the left ventricular 5-HT2BR overexpression, 5-HT2BR blockade by RS-127445 did not affect left ventricular hypertrophy and fibrosis in SHRs. This antagonist did not improve diastolic dysfunction, neither alone nor in combination with nicardipine, although it induced plasma brain natriuretic peptide decrease. Moreover, RS-127445 amplified subendocardial fibrosis and favored left ventricular dilatation. Finally, a subendocardial left ventricular fibrosis was induced by chronic serotonin in wild-type mice, which was increased in Htr2B animals, but prevented in Htr2A and Htr2A/2B mice, and could be explained by a contribution of the endothelial 5-HT2BRs to coronary vasodilatation. CONCLUSION: This work is the first to identify a cardioprotective function of the 5-HT2BR in an integrated model of diastolic dysfunction with preserved ejection fraction.
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Presión Sanguínea/efectos de los fármacos , Hipertensión/metabolismo , Hipertrofia Ventricular Izquierda/metabolismo , Antagonistas de la Serotonina/farmacología , Serotonina/metabolismo , Disfunción Ventricular Izquierda/metabolismo , Animales , Presión Sanguínea/fisiología , Ecocardiografía , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/fisiopatología , Hipertensión/fisiopatología , Hipertrofia Ventricular Izquierda/fisiopatología , Masculino , Ratones , Ratones Noqueados , Péptido Natriurético Encefálico/metabolismo , Pirimidinas/farmacología , Ratas , Ratas Endogámicas SHR , Receptor de Serotonina 5-HT2A/genética , Receptor de Serotonina 5-HT2A/metabolismo , Receptor de Serotonina 5-HT2B/genética , Receptor de Serotonina 5-HT2B/metabolismo , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
Partial monosomy 21 (PM21) is a rare chromosomal abnormality that is characterized by the loss of a variable segment along human chromosome 21 (Hsa21). The clinical phenotypes of this loss are heterogeneous and range from mild alterations to lethal consequences, depending on the affected region of Hsa21. The most common features include intellectual disabilities, craniofacial dysmorphology, short stature, and muscular and cardiac defects. As a complement to human genetic approaches, our team has developed new monosomic mouse models that carry deletions on Hsa21 syntenic regions in order to identify the dosage-sensitive genes that are responsible for the symptoms. We focus here on the Ms5Yah mouse model, in which a 7.7-Mb region has been deleted from the App to Runx1 genes. Ms5Yah mice display high postnatal lethality, with a few surviving individuals showing growth retardation, motor coordination deficits, and spatial learning and memory impairments. Further studies confirmed a gene dosage effect in the Ms5Yah hippocampus, and pinpointed disruptions of pathways related to cell adhesion (involving App, Cntnap5b, Lgals3bp, Mag, Mcam, Npnt, Pcdhb2, Pcdhb3, Pcdhb4, Pcdhb6, Pcdhb7, Pcdhb8, Pcdhb16 and Vwf). Our PM21 mouse model is the first to display morphological abnormalities and behavioural phenotypes similar to those found in affected humans, and it therefore demonstrates the major contribution that the App-Runx1 region has in the pathophysiology of PM21.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Monosomía/genética , Eliminación de Secuencia/genética , Animales , Animales Recién Nacidos , Conducta Animal , Peso Corporal , Cromosomas Humanos Par 21/genética , Análisis por Conglomerados , Subunidad alfa 2 del Factor de Unión al Sitio Principal/deficiencia , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Modelos Animales de Enfermedad , Conducta Exploratoria , Feto/anomalías , Feto/patología , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Aprendizaje por Laberinto , Memoria , Ratones , Anotación de Secuencia Molecular , Actividad Motora , Análisis de Secuencia por Matrices de Oligonucleótidos , Programas Informáticos , Aprendizaje Espacial , Transcriptoma/genéticaRESUMEN
Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.
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Desarrollo Embrionario/genética , Gastrulación/genética , Proteína de Unión al Tracto de Polipirimidina/fisiología , Animales , Blastocisto/metabolismo , Blastocisto/fisiología , Diferenciación Celular/genética , División Celular/genética , Embrión de Mamíferos , Femenino , Gastrulación/fisiología , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Proteína de Unión al Tracto de Polipirimidina/genética , Embarazo , Factores de TiempoRESUMEN
TIF1ß is an essential mammalian transcriptional corepressor. It interacts with the heterochromatin proteins HP1 through a highly conserved motif, the HP1box, and we have previously shown that this interaction is essential for the differentiation of F9 cells to occur. Here we address the in vivo functions of the TIF1ß-HP1 interaction, by generating mice in which the TIF1ß HP1box is mutated, leading to the loss of TIF1ß interaction with HP1. The effects of the mutation were monitored in two instances, where TIF1ß is known to play key roles: early embryonic development and spermatogenesis. We find that mutating the HP1box of TIF1ß disrupts embryonic development soon after gastrulation. This effect is likely caused by the misexpression of TIF1ß targets that regulate mitotic progression and pluripotency. In contrast, in Sertoli cells, we found that the absence of TIF1ß but not its mutation in the HP1box leads to a clear defect of spermatogenesis characterized by a failure of spermatid release and a testicular degeneration. These data show that the interaction between TIF1ß and HP1 is essential for some but not all TIF1ß functions in vivo. Furthermore, we observed that TIF1ß is dispersed through the nucleoplasm of E7.0 embryos, whereas it is mainly associated with pericentromeric heterochromatin of E8.5 embryos and of Sertoli cells, an association that is lost upon TIF1ß HP1box mutation. Altogether, these data provide strong evidence that nuclear organization plays key roles during early embryonic development.
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Proteínas Cromosómicas no Histona/fisiología , Gastrulación , Proteínas Nucleares/fisiología , Proteínas Represoras/fisiología , Células de Sertoli/fisiología , Espermatogénesis , Animales , Ciclo Celular , Homólogo de la Proteína Chromobox 5 , Desarrollo Embrionario , Femenino , Proteínas de Homeodominio/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Homeótica Nanog , Proteínas Nucleares/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Proteínas Represoras/genética , Proteína 28 que Contiene Motivos TripartitoRESUMEN
Conditional gene manipulations in mice are increasingly popular strategies in biomedical research. These approaches rely on the production of conditional genetically engineered mutant mouse (GEMM) lines with mutations in protein-encoding genes. These conditional GEMMs are then bred with one or several transgenic mouse lines expressing a site-specific recombinase, most often the Cre recombinase, in a tissue-specific manner. Conditional GEMMs can only be exploited if Cre transgenic mouse lines are available to generate somatic mutations, and thus the number of Cre transgenic lines has significantly increased over the last 15 years. Once produced, these transgenic lines must be validated for reliable, efficient, and specific Cre expression and Cre-mediated recombination. In this overview, the minimum level of information that is ideally required to validate a Cre-driver transgenic line is first discussed. The vagaries associated with validation procedures are considered next, and some solutions are proposed to assess the expression and activity of constitutive or inducible Cre recombinase before undertaking extensive breeding experiments and exhaustive phenotyping. Curr. Protoc. Mouse Biol. 1:1-15. © 2011 by John Wiley & Sons, Inc.
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A procedure for post-mortem examination (or necropsy) of mice is provided. The aim is to obtain a "holistic" picture of organs and systems at the anatomical and histological levels. The major issue is tissue preservation, which is achieved by rapid transfer into a fixative solution, usually neutral buffered formalin. Fixation is the first of the four basic steps in histopathological analyses of tissues, which also include embedding, sectioning, and staining. The protocols provided here describe routine methods for tissue fixation, as these methods are integral parts of any necropsy procedure. There is also a Strategic Planning section that addresses the overall approach to histopathological evaluation, as well as specifics such as age and gender of the mice, cohort size, and controls. Curr. Protoc. Mouse Biol. 1:17-53. © 2011 by John Wiley & Sons, Inc.
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The definition of embryonic potency and induction of specific cell fates are intimately linked to the tight control over TGFbeta signaling. Although extracellular regulation of ligand availability has received considerable attention in recent years, surprisingly little is known about the intracellular factors that negatively control Smad activity in mammalian tissues. By means of genetic ablation, we show that the Smad4 inhibitor ectodermin (Ecto, also known as Trim33 or Tif1gamma) is required to limit Nodal responsiveness in vivo. New phenotypes, which are linked to excessive Nodal activity, emerge from such a modified landscape of Smad responsiveness in both embryonic and extra-embryonic territories. In extra-embryonic endoderm, Ecto is required to confine expression of Nodal antagonists to the anterior visceral endoderm. In trophoblast cells, Ecto precisely doses Nodal activity, balancing stem cell self-renewal and differentiation. Epiblast-specific Ecto deficiency shifts mesoderm fates towards node/organizer fates, revealing the requirement of Smad inhibition for the precise allocation of cells along the primitive streak. This study unveils that intracellular negative control of Smad function by ectodermin/Tif1gamma is a crucial element in the cellular response to TGFbeta signals in mammalian tissues.
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Regulación del Desarrollo de la Expresión Génica , Proteínas Smad/metabolismo , Factores de Transcripción/metabolismo , Alelos , Animales , Tipificación del Cuerpo , Diferenciación Celular , Cruzamientos Genéticos , Ectodermo/metabolismo , Mesodermo/metabolismo , Ratones , Modelos Biológicos , Fenotipo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
To generate temporally-controlled targeted somatic mutations selectively and efficiently in smooth muscles, we have established a transgenic SMA-Cre-ER(T2) mouse line in which the expression of the Tamoxifen-dependent Cre-ER(T2) recombinase is under the control of a large genomic DNA segment of the mouse smooth muscle alpha actin (SMA) gene, contained in a Bacterial artificial chromosome (Bac). In this transgenic mouse line, Cre-ER(T2)-mediated recombination of LoxP-flanked target DNA is strictly Tamoxifen-dependent, and efficient in both vascular and visceral smooth muscle cells. Moreover, with the exception of few cardiomyocytes, LoxP-flanked DNA excision is restricted to smooth muscle cells. Thus, SMA-Cre-ER(T2) mice should be of great value to analyze gene function in smooth muscles, and to establish new animal models of human smooth muscle disorders.
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Envejecimiento/fisiología , Ingeniería Genética/métodos , Mutagénesis/genética , Miocitos del Músculo Liso/metabolismo , Animales , Ratones , Ratones Transgénicos , Factores de TiempoRESUMEN
Sirtuins (SIRTs) are class-III NAD-dependent histone deacetylases (HDACs) that regulate various physiological processes. Inactivation of SIRT1 in the mouse leads to male sterility, but the molecular mechanisms responsible for this phenotype have not been determined. Here we show that fetal testis development appears normal in Sirt1(-/-) mice. In contrast, the first round of spermatogenesis arrests before the completion of meiosis with abundant apoptosis of pachytene spermatocytes, abnormal Leydig and Sertoli cell maturation, and strongly reduced intratesticular testosterone levels. We show that this phenotype is the consequence of diminished hypothalamic gonadotropin-releasing hormone expression and strongly reduced luteinizing hormone levels. Rather than having an intrinsic effect on male germ cells per se, our results show that SIRT1 regulates spermatogenesis at postnatal stages by controlling hypothalamus-pituitary gonadotropin (HPG) signaling. In addition to its well studied role in control of metabolism and energy homeostasis, our results thus reveal a novel and critical function of SIRT1 in controlling HPG signaling. This phenotype is more severe than those previously described using mice bred on different genetic backgrounds, and highlights the fact that SIRT1 function is strongly modified by other genetic loci.
Asunto(s)
Fertilidad/genética , Gonadotropinas Hipofisarias/fisiología , Sistema Hipotálamo-Hipofisario/fisiopatología , Infertilidad Masculina/genética , Sirtuinas/fisiología , Animales , Diferenciación Celular/genética , Fertilidad/fisiología , Gonadotropinas Hipofisarias/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/fisiología , Sistema Hipotálamo-Hipofisario/metabolismo , Infertilidad Masculina/fisiopatología , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/fisiología , Masculino , Ratones , Ratones Noqueados , Células de Sertoli/metabolismo , Células de Sertoli/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Sirtuina 1 , Sirtuinas/genética , Espermatogénesis/genética , Testosterona/biosíntesisRESUMEN
In this unit, a procedure for post-mortem examination of mice and tissue collection is provided. This procedure is performed for post-mortem analysis of anatomical defects (necropsy) and histological analysis and/or tissue collection destined for molecular biology applications. In both cases, tissue preservation is the major issue, but the way to achieve it depends on the objective. When histological analysis is the aim, tissue preservation is achieved by rapid transfer into fixative solutions. In contrast, molecular biology applications require rapid freezing of tissue samples to preserve mRNA integrity. Consequently, performing both procedures simultaneously may be at the expense of the final product quality.