RESUMEN
Purpose: The clinical phenotype of retinal gliosis occurs in different forms; here, we characterize one novel genetic feature, (i.e., signaling via BMP-receptor 1b). Methods: Mouse mutants were generated within a recessive ENU mutagenesis screen; the underlying mutation was identified by linkage analysis and Sanger sequencing. The eye phenotype was characterized by fundoscopy, optical coherence tomography, optokinetic drum, electroretinography, and visual evoked potentials, by histology, immunohistology, and electron-microscopy. Results: The mutation affects intron 10 of the Bmpr1b gene, which is causative for skipping of exon 10. The expression levels of pSMAD1/5/8 were reduced in the mutant retina. The loss of BMPR1B-mediated signaling leads to optic nerve coloboma, gliosis in the optic nerve head and ventral retina, defective optic nerve axons, and irregular retinal vessels. The ventral retinal gliosis is proliferative and hypertrophic, which is concomitant with neuronal delamination and the reduction of retinal ganglion cells (RGCs); it is dominated by activated astrocytes overexpressing PAX2 and SOX2 but not PAX6, indicating that they may retain properties of gliogenic precursor cells. The expression pattern of PAX2 in the optic nerve head and ventral retina is altered during embryonic development. These events finally result in reduced electrical transmission of the retina and optic nerve and significantly reduced visual acuity. Conclusions: Our study demonstrates that BMPR1B is necessary for the development of the optic nerve and ventral retina. This study could also indicate a new mechanism in the formation of retinal gliosis; it opens new routes for its treatment eventually preventing scar formation in the retina.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Coloboma/genética , Gliosis/genética , Mutación , Disco Óptico/anomalías , Enfermedades de la Retina/genética , Animales , Ratones , Disco Óptico/patologíaRESUMEN
BACKGROUND: Syndromic forms of osteosarcoma (OS) account for less than 10% of all recorded cases of this malignancy. An individual OS predisposition is also possible by the inheritance of low penetrance alleles of tumor susceptibility genes, usually without evidence of a syndromic condition. Genetic variants involved in such a non-syndromic form of tumor predisposition are difficult to identify, given the low incidence of osteosarcoma cases and the genetic heterogeneity of patients. We recently mapped a major OS susceptibility QTL to mouse chromosome 14 by comparing alpha-radiation induced osteosarcoma in mouse strains which differ in their tumor susceptibility. METHODS: Tumor-specific allelic losses in murine osteosacoma were mapped along chromosome 14 using microsatellite markers and SNP allelotyping. Candidate gene search in the mapped interval was refined using PosMed data mining and mRNA expression analysis in normal osteoblasts. A strain-specific promoter variant in Rb1 was tested for its influence on mRNA expression using reporter assay. RESULTS: A common Rb1 allele derived from the BALB/cHeNhg strain was identified as the major determinant of radiation-induced OS risk at this locus. Increased OS-risk is linked with a hexanucleotide deletion in the promoter region which is predicted to change WT1 and SP1 transcription factor-binding sites. Both in-vitro reporter and in-vivo expression assays confirmed an approx. 1.5 fold reduced gene expression by this promoter variant. Concordantly, the 50% reduction in Rb1 expression in mice bearing a conditional hemizygous Rb1 deletion causes a significant rise of OS incidence following alpha-irradiation. CONCLUSION: This is the first experimental demonstration of a functional and genetic link between reduced Rb1 expression from a common promoter variant and increased tumor risk after radiation exposure. We propose that a reduced Rb1 expression by common variants in regulatory regions can modify the risk for a malignant transformation of bone cells after radiation exposure.
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Predisposición Genética a la Enfermedad , Variación Genética , Osteosarcoma/genética , Osteosarcoma/patología , Regiones Promotoras Genéticas , Radiación , Proteína de Retinoblastoma/genética , Regiones no Traducidas 3'/genética , Desequilibrio Alélico , Animales , Secuencia de Bases , Sitios de Unión , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Cromosomas de los Mamíferos/genética , Cruzamientos Genéticos , Femenino , Regulación Neoplásica de la Expresión Génica , Estudios de Asociación Genética , Humanos , Hibridación Genética , Mutación INDEL/genética , Masculino , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de RiesgoRESUMEN
BACKGROUND: Type I Bartter syndrome is a recessive human nephropathy caused by loss-of-function mutations in the SLC12A1 gene coding for the Na+-K+-2Cl- cotransporter NKCC2. We recently established the mutant mouse line Slc12a1I299F exhibiting kidney defects highly similar to the late-onset manifestation of this hereditary human disease. Besides the kidney defects, low blood pressure and osteopenia were revealed in the homozygous mutant mice which were also described in humans. Beside its strong expression in the kidney, NKCC2 has been also shown to be expressed in other tissues in rodents i.e. the gastrointestinal tract, pancreatic beta cells, and specific compartments of the ear, nasal tissue and eye. RESULTS: To examine if, besides kidney defects, further organ systems and/or metabolic pathways are affected by the Slc12a1I299F mutation as primary or secondary effects, we describe a standardized, systemic phenotypic analysis of the mutant mouse line Slc12a1I299F in the German Mouse Clinic. Slc12a1I299F homozygous mutant mice and Slc12a1I299F heterozygous mutant littermates as controls were tested at the age of 4-6 months. Beside the already published changes in blood pressure and bone metabolism, a significantly lower body weight and fat content were found as new phenotypes for Slc12a1I299F homozygous mutant mice. Small additional effects included a mild erythropenic anemia in homozygous mutant males as well as a slight hyperalgesia in homozygous mutant females. For other functions, such as immunology, lung function and neurology, no distinct alterations were observed. CONCLUSIONS: In this systemic analysis no clear primary effects of the Slc12a1I299F mutation appeared for the organs other than the kidneys where Slc12a1 expression has been described. On the other hand, long-term effects additional and/or secondary to the kidney lesions might also appear in humans harboring SLC12A1 mutations.
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Síndrome de Bartter , Presión Sanguínea/genética , Mutación Missense , Miembro 1 de la Familia de Transportadores de Soluto 12 , Sustitución de Aminoácidos , Animales , Síndrome de Bartter/genética , Síndrome de Bartter/metabolismo , Síndrome de Bartter/patología , Huesos/metabolismo , Huesos/patología , Femenino , Homocigoto , Humanos , Hiperalgesia/genética , Hiperalgesia/metabolismo , Hiperalgesia/patología , Masculino , Ratones , Ratones Mutantes , Miembro 1 de la Familia de Transportadores de Soluto 12/genética , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
Collagen type IV alpha 1 and 2 (COL4A1 and COL4A2) are present in nearly all basement membranes. COL4A1 and COL4A2 mutations are pleiotropic, affecting multiple organ systems to differing degrees, and both genetic-context and environmental factors influence this variable expressivity. Here, we report important phenotypic and molecular differences in an allelic series of Col4a1 and Col4a2 mutant mice that are on a uniform genetic background. We evaluated three organs commonly affected by COL4A1 and COL4A2 mutations and discovered allelic heterogeneity in the penetrance and severity of ocular dysgenesis, myopathy and brain malformations. Similarly, we show allelic heterogeneity in COL4A1 and COL4A2 biosynthesis. While most mutations that we examined caused increased intracellular and decreased extracellular COL4A1 and COL4A2, we identified three mutations with distinct biosynthetic signatures. Reduced temperature or presence of 4-phenylbutyrate ameliorated biosynthetic defects in primary cell lines derived from mutant mice. Together, our data demonstrate the effects and clinical implications of allelic heterogeneity in Col4a1- and Col4a2-related diseases. Understanding allelic differences will be valuable for increasing prognostic accuracy and for the development of therapeutic interventions that consider the nature of the molecular cause in patients with COL4A1 and COL4A2 mutations.
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Encéfalo/anomalías , Colágeno Tipo IV/genética , Anomalías del Ojo/genética , Enfermedades Musculares/genética , Enfermedades del Nervio Óptico/congénito , Alelos , Animales , Antineoplásicos/farmacología , Membrana Basal/metabolismo , Células Cultivadas , Estrés del Retículo Endoplásmico/genética , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Enfermedades del Nervio Óptico/genética , Penetrancia , Fenilbutiratos/farmacología , Pliegue de Proteína , TemperaturaRESUMEN
Cln3(Δex7/8) mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3(Δex7/8) mice. Homozygous Cln3(Δex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Δex7/8) mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12-13 week old homozygous Cln3(Δex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Δex7/8) mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15-16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3(Δ) (ex7/8) mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3(Δ) (ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Δ) (ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Δ) (ex7/8) mice that merit further study for JNCL biomarker development.
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Modelos Animales de Enfermedad , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Lipofuscinosis Ceroideas Neuronales/patología , Fenotipo , Degeneración Retiniana/patología , Análisis de Varianza , Animales , Temperatura Corporal , Encéfalo/patología , Electrorretinografía , Conducta Exploratoria/fisiología , Femenino , Ferritinas/sangre , Genotipo , Corazón/crecimiento & desarrollo , Inmunohistoquímica , Linfocitos/patología , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Chaperonas Moleculares/genética , Lipofuscinosis Ceroideas Neuronales/complicaciones , Lipofuscinosis Ceroideas Neuronales/genética , Lipofuscinosis Ceroideas Neuronales/metabolismo , Tamaño de los Órganos , Consumo de Oxígeno/fisiología , Degeneración Retiniana/etiologíaRESUMEN
Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (-50 and -29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced P(enh) and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (-12%), reduced total oxygen consumption rate (-8%), improved glucose tolerance, and reduced grip force (-14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long-term lung pathology develops in the knockout mice due to impairment of energy-costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma.
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Complejo IV de Transporte de Electrones/metabolismo , Pulmón/patología , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Cartilla de ADN , Complejo IV de Transporte de Electrones/genética , Pulmón/enzimología , Pulmón/fisiología , Ratones , Ratones Noqueados , Reacción en Cadena de la PolimerasaRESUMEN
Metabolic bone disorders arise as primary diseases or may be secondary due to a multitude of organ malfunctions. Animal models are required to understand the molecular mechanisms responsible for the imbalances of bone metabolism in disturbed bone mineralization diseases. Here we present the isolation of mutant mouse models for metabolic bone diseases by phenotyping blood parameters that target bone turnover within the large-scale genome-wide Munich ENU Mutagenesis Project. A screening panel of three clinical parameters, also commonly used as biochemical markers in patients with metabolic bone diseases, was chosen. Total alkaline phosphatase activity and total calcium and inorganic phosphate levels in plasma samples of F1 offspring produced from ENU-mutagenized C3HeB/FeJ male mice were measured. Screening of 9,540 mice led to the identification of 257 phenodeviants of which 190 were tested by genetic confirmation crosses. Seventy-one new dominant mutant lines showing alterations of at least one of the biochemical parameters of interest were confirmed. Fifteen mutations among three genes (Phex, Casr, and Alpl) have been identified by positional-candidate gene approaches and one mutation of the Asgr1 gene, which was identified by next-generation sequencing. All new mutant mouse lines are offered as a resource for the scientific community.
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Enfermedades Óseas Metabólicas/genética , Modelos Animales de Enfermedad , Ratones/genética , Fosfatasa Alcalina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Enfermedades Óseas Metabólicas/sangre , Enfermedades Óseas Metabólicas/enzimología , Calcio/sangre , Cromosomas de los Mamíferos , Análisis Mutacional de ADN , Etilnitrosourea/farmacología , Femenino , Masculino , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Mutagénesis , Mutágenos/farmacología , Mutación , Endopeptidasa Neutra Reguladora de Fosfato PHEX/genética , Fenotipo , Fosfatos/sangre , Polimorfismo de Nucleótido Simple , Receptores Sensibles al Calcio/genética , Estadísticas no Paramétricas , Cromosoma XRESUMEN
Collagen, type IV, alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers and are abundant components of basement membranes, including those of the cerebral vasculature. COL4A1 mutations are an increasingly recognized cause of multisystem disorders, including highly penetrant cerebrovascular disease and intracerebral hemorrhage (ICH). Because COL4A1 and COL4A2 are structurally and functionally associated, we hypothesized that variants in COL4A2 would also cause ICH. We sequence COL4A2 in 96 patients with ICH and identify three rare, nonsynonymous coding variants in four patients that are not present in a cohort of 144 ICH-free individuals. All three variants change evolutionarily conserved amino acids. Using a cellular assay, we show that these putative mutations cause intracellular accumulation of COL4A1 and COL4A2 at the expense of their secretion, which supports their pathogenecity. Furthermore, we show that Col4a2 mutant mice also have completely penetrant ICH and that mutations in mouse and human lead to retention of COL4A1 and COL4A2 within the endoplasmic reticulum (ER). Importantly, two of the three putative mutations found in patients trigger ER stress and activate the unfolded protein response. The identification of putative COL4A2 mutations that might contribute to ICH in human patients provides insight into the pathogenic mechanisms of this disease. Our data suggest that COL4A2 mutations impair COL4A1 and COL4A2 secretion and can also result in cytotoxicity. Finally, our findings suggest that, collectively, mutations in COL4A1 and COL4A2 contribute to sporadic cases of ICH.
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Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Hemorragias Intracraneales/genética , Mutación , Accidente Cerebrovascular/genética , Adulto , Anciano , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Estrés del Retículo Endoplásmico/genética , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Datos de Secuencia Molecular , Respuesta de Proteína DesplegadaRESUMEN
Glial cell line-derived neurotrophic factor (GDNF) enhances the survival of a variety of neurons, including photoreceptors (PR) in the retina. In contrast to most other GDNF receptive neurons, GDNF does, however, not exert its neuroprotective activity directly on PR neurons but transmits it indirectly by inducing expression of yet unknown neurotrophic factors in retinal Müller glial (RMG) cells. Genome-wide differential transcriptome analyses of GDNF-treated mouse retinas revealed 30 GDNF-induced transcripts containing a total of six genes coding for secreted molecules. Among them was (OPN), a secreted glycoprotein which was expressed in mouse RMG and secreted from primary mouse RMG in culture. Furthermore, OPN secretion was significantly upregulated on GDNF treatment of primary RMG. To validate, whether OPN could qualify as a neuroprotective factor for PR, we evaluated its potential neurotrophic activity on isolated PR in vitro as well as on retinal explants from the retinal degeneration 1 (Pde6brd1) mouse mutant. OPN exerted a significant, positive survival effect on primary porcine PR cells in a concentration-dependent manner and induced activation of PI3K/Akt pro-survival pathway. Moreover, in retinal explant cultures from Pde6brd1 mice, OPN significantly reduced the percentage of apoptotic cells to levels comparable with that observed in explants from wild-type mice and led to survival of significantly more PR in long-term retinal explant cultures. Our findings suggest that RMG-derived OPN is a novel candidate protein that transmits part of the GDNF-induced neuroprotective activity of RMG to PR cells.
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Supervivencia Celular/efectos de los fármacos , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Neuroglía/metabolismo , Osteopontina/farmacología , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Degeneración Retiniana/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Western Blotting , Supervivencia Celular/fisiología , Ensayo de Inmunoadsorción Enzimática , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Ratones Transgénicos , Neuroglía/efectos de los fármacos , Osteopontina/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismoAsunto(s)
Genética/historia , Alemania , Historia del Siglo XX , Historia del Siglo XXI , Estados UnidosRESUMEN
Renal-coloboma syndrome, also known as papillorenal syndrome, is an autosomal dominant human disorder in which optic disc coloboma is associated with kidney abnormalities. Mutations in the paired domain transcription factor PAX2 have been found to be the underlying cause of this disease. Disease severity varies between patients, and in some cases, renal hypoplasia has been found in the absence of any retinal defects. Here we report an N-ethyl-N-nitrosourea-induced mouse mutation, Opdc, which is an isoleucinetothreonine missense mutation, I40T, in the first α-helix of the Pax2 paired domain. The mutant protein binds target DNA sequences less strongly than the wild-type protein and acts poorly to transactivate target promoters in culture. The phenotypic consequence of this mutation on the development of the eye and ear is similar to that reported for null alleles of Pax2. However, in homozygotes, cerebellar development is normal on a genetic background in which loss of Pax2 results in failure of cerebellar formation. Moreover, there is a genetic background effect on the heterozygous phenotype such that on some strain backgrounds, kidney development is unaffected. Opdc is the first hypomorphic mutation reported for Pax2 that differs in phenotype from loss-of-function mutations. These results suggest that PAX2 is a strong candidate gene for cases in which human patients have optic disc coloboma not associated with renal dysplasia.
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Coloboma/genética , Coloboma/patología , Mutación Missense , Factor de Transcripción PAX2/genética , Factor de Transcripción PAX2/metabolismo , Fenotipo , Insuficiencia Renal/genética , Insuficiencia Renal/patología , Reflujo Vesicoureteral/genética , Reflujo Vesicoureteral/patología , Anomalías Múltiples/genética , Anomalías Múltiples/patología , Animales , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Genotipo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Modelos Animales , Mutación Puntual , Activación Transcripcional/genéticaRESUMEN
Most neurons in the adult mammalian brain survive for the entire life of an individual. However, it is not known which transcriptional pathways regulate this survival in a healthy brain. Here, we identify a pathway regulating neuronal survival in a highly subtype-specific manner. We show that the transcription factor Pax6 expressed in dopaminergic neurons of the olfactory bulb regulates the survival of these neurons by directly controlling the expression of crystallin αA (CryαA), which blocks apoptosis by inhibition of procaspase-3 activation. Re-expression of CryαA fully rescues survival of Pax6-deficient dopaminergic interneurons in vivo and knockdown of CryαA by shRNA in wild-type mice reduces the number of dopaminergic OB interneurons. Strikingly, Pax6 utilizes different DNA-binding domains for its well-known role in fate specification and this role of regulating the survival of specific neuronal subtypes in the mature, healthy brain.
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Cristalinas/fisiología , Dopamina/fisiología , Proteínas del Ojo/fisiología , Proteínas de Homeodominio/fisiología , Neuronas/fisiología , Bulbo Olfatorio/fisiología , Factores de Transcripción Paired Box/fisiología , Proteínas Represoras/fisiología , Animales , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Cristalinas/genética , Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/citología , Neuronas/metabolismo , Bulbo Olfatorio/citología , Bulbo Olfatorio/metabolismo , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Proteínas Represoras/genéticaRESUMEN
BACKGROUND: The transcription factor Pax6 functions in the specification and maintenance of the differentiated cell lineages in the endocrine pancreas. It has two DNA binding domains, the paired domain and the homeodomain, in addition to a C-terminal transactivation domain. The phenotype of Pax6-/- knockout mice suggests non-redundant functions of the transcription factor in the development of glucagon-expressing alpha-cells as this cell type is absent in the mutants. We ask the question of how the differentiation of pancreatic endocrine cells, in particular that of alpha-cells, is affected by selective inactivation of either one of the three major domains of Pax6. RESULTS: The Pax6Aey18 mutant mouse line, in which the paired domain is inactivated, showed a phenotype similar to that of Pax6-/- knockout mice with a near complete absence of glucagon-positive alpha-cells (0-4 cells/section; < or =1% of wt), reduced beta-cell area (74% of wt) and disorganized islets. The proportion of ghrelin-positive epsilon-cells was expanded. In Pax6Sey-Neu mutants, which lack the transactivation domain, alpha-and beta-cells where reduced to 25 and 40% of wt, respectively. We also studied two mouse lines with mutations in the homeodomain, Pax64Neu and Pax6132-14Neu. Neighboring amino acids are affected in the two lines and both point mutations abolish DNA binding of the classical P3 homeodomain target sequence. The pancreatic phenotype of the two mutants however was divergent. While Pax64Neu homozygotes showed a reduction of alpha- and beta-cells to 59 and 61%, respectively, pancreatic endocrine development was unaltered in the Pax6132-14Neu mutant strain. CONCLUSIONS: We show that inactivation of the Pax6 paired domain leads to a more severe phenotype with regards to the differentiation of pancreatic alpha-cells than the loss of the transactivation domain. The analysis of two different homeodomain mutants suggests that the binding of Pax6 to P3 homeodomain consensus sequences is not required for alpha-cell development. It rather seems that the homeodomain has a modulating role in Pax6 function, possibly by facilitating a PH0-like binding confirmation on paired domain target genes like proglucagon. This function is differentially affected by the two homeodomain mutations analyzed in this study.
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Proteínas del Ojo/metabolismo , Células Secretoras de Glucagón/citología , Proteínas de Homeodominio/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Animales , Proteínas del Ojo/química , Proteínas de Homeodominio/química , Ratones , Ratones Noqueados , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/química , Páncreas/citología , Páncreas/embriología , Estructura Terciaria de Proteína , Proteínas Represoras/químicaRESUMEN
A new spontaneous mouse mutant was characterized by closed eyelids at weaning and without apparent eyes (provisional gene name, eyeless; provisional gene symbol, eyl). The mutation follows a recessive pattern of inheritance and was mapped to the region of chromosome 19 containing Pitx3. Genetic complementation tests using Pitx3 ( ak/+ ) mice confirmed eyl as a new allele of Pitx3 (Pitx3 ( eyl )). Sequencing of the Pitx3 gene in eyl mutants identified an inserted G after cDNA position 416 (416insG; exon 4). The shifted open reading frame is predicted to result in a hybrid protein still containing the Pitx3 homeobox, but followed by 121 new amino acids. The novel Pitx3 ( eyl/eyl ) mutants expressed ophthalmological and brain defects similar to Pitx3 ( ak/ak ) mice: microphthalmia or anophthalmia and loss of dopamine neurons of the substantia nigra. In addition, we observed in the homozygous eyeless mutants increased extramedullary hematopoiesis in the spleen, frequently liver steatosis, and reduced body weight. There were also several behavioral changes in the homozygous mutants, including reduced forelimb grip strength and increased nociception. In addition to these alterations in both sexes, we observed in female Pitx3 ( eyl/eyl ) mice increased anxiety-related behavior, reduced locomotor activity, reduced object exploration, and increased social contacts; however, we observed decreased anxiety-related behavior and increased arousal in males. Most of these defects identified in the new Pitx3 mutation are observed in Parkinson patients, making the Pitx3 ( eyl ) mutant a valuable new model. It is the first mouse mutant carrying a point mutation within the coding region of Pitx3.
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Ratones Mutantes/genética , Microftalmía/genética , Dolor/genética , Trastornos Parkinsonianos/genética , Secuencia de Aminoácidos , Animales , Anoftalmos/genética , Secuencia de Bases , Conducta Animal , Densidad Ósea , Enfermedades Óseas/genética , Enfermedades Óseas/fisiopatología , Mapeo Cromosómico , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Hígado Graso/genética , Hígado Graso/fisiopatología , Femenino , Proteínas de Homeodominio/genética , Enfermedades Pulmonares/genética , Enfermedades Pulmonares/fisiopatología , Masculino , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Mutación Puntual , Tirosina 3-Monooxigenasa/genéticaRESUMEN
It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution due to effects on aspects of speech and language. Here, we introduce these substitutions into the endogenous Foxp2 gene of mice. Although these mice are generally healthy, they have qualitatively different ultrasonic vocalizations, decreased exploratory behavior and decreased dopamine concentrations in the brain suggesting that the humanized Foxp2 allele affects basal ganglia. In the striatum, a part of the basal ganglia affected in humans with a speech deficit due to a nonfunctional FOXP2 allele, we find that medium spiny neurons have increased dendrite lengths and increased synaptic plasticity. Since mice carrying one nonfunctional Foxp2 allele show opposite effects, this suggests that alterations in cortico-basal ganglia circuits might have been important for the evolution of speech and language in humans.
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Sustitución de Aminoácidos , Ganglios Basales/metabolismo , Evolución Biológica , Factores de Transcripción Forkhead/metabolismo , Vocalización Animal , Animales , Dendritas/metabolismo , Dopamina/metabolismo , Expresión Génica , Heterocigoto , Humanos , Lenguaje , Depresión Sináptica a Largo Plazo , Ratones , Vías Nerviosas , Plasticidad Neuronal , HablaRESUMEN
In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different from intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca(+2)-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions.
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Proteínas de Unión al Calcio/genética , Anomalías del Ojo/genética , Proteínas del Ojo/genética , Eliminación de Gen , Proteínas de Homeodominio/genética , Factores de Transcripción Paired Box/genética , Proteínas Represoras/genética , Animales , Tipificación del Cuerpo/genética , Proteínas de Unión al Calcio/fisiología , Proteínas del Ojo/fisiología , Genes Letales , Proteínas de Homeodominio/fisiología , Ratones , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/fisiología , Fenotipo , Proteínas Represoras/fisiologíaRESUMEN
With the completion of the mouse genome sequence an essential task for biomedical sciences in the twenty-first century will be the generation and functional analysis of mouse models for every gene in the mammalian genome. More than 30,000 mutations in ES cells will be engineered and thousands of mouse disease models will become available over the coming years by the collaborative effort of the International Mouse Knockout Consortium. In order to realize the full value of the mouse models proper characterization, archiving and dissemination of mouse disease models to the research community have to be performed. Phenotyping centers (mouse clinics) provide the necessary capacity, broad expertise, equipment, and infrastructure to carry out large-scale systemic first-line phenotyping. Using the example of the German Mouse Clinic (GMC) we will introduce the reader to the different aspects of the organization of a mouse clinic and present selected methods used in first-line phenotyping.
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Almacenamiento y Recuperación de la Información/métodos , Fenotipo , Animales , Sistemas de Administración de Bases de Datos , RatonesRESUMEN
BACKGROUND: In Drosophila, mutations in the gene eyes absent (eya) lead to severe defects in eye development. The functions of its mammalian orthologs Eya1-4 are only partially understood and no mouse model exists for Eya3. Therefore, we characterized the phenotype of a new Eya3 knockout mouse mutant. RESULTS: Expression analysis of Eya3 by in-situ hybridizations and beta-Gal-staining of Eya3 mutant mice revealed abundant expression of the gene throughout development, e.g. in brain, eyes, heart, somites and limbs suggesting pleiotropic effects of the mutated gene. A similar complex expression pattern was observed also in zebrafish embryos. The phenotype of young adult Eya3 mouse mutants was systematically analyzed within the German Mouse Clinic. There was no obvious defect in the eyes, ears and kidneys of Eya3 mutant mice. Homozygous mutants displayed decreased bone mineral content and shorter body length. In the lung, the tidal volume at rest was decreased, and electrocardiography showed increased JT- and PQ intervals as well as decreased QRS amplitude. Behavioral analysis of the mutants demonstrated a mild increase in exploratory behavior, but decreased locomotor activity and reduced muscle strength. Analysis of differential gene expression revealed 110 regulated genes in heart and brain. Using real-time PCR, we confirmed Nup155 being down regulated in both organs. CONCLUSION: The loss of Eya3 in the mouse has no apparent effect on eye development. The wide-spread expression of Eya3 in mouse and zebrafish embryos is in contrast to the restricted expression pattern in Xenopus embryos. The loss of Eya3 in mice leads to a broad spectrum of minor physiological changes. Among them, the mutant mice move less than the wild-type mice and, together with the effects on respiratory, muscle and heart function, the mutation might lead to more severe effects when the mice become older. Therefore, future investigations of Eya3 function should focus on aging mice.
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Proteínas de Unión al ADN/deficiencia , Animales , Secuencia de Bases , ADN/genética , Proteínas de Unión al ADN/genética , Ojo/embriología , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Homocigoto , Hibridación in Situ , Operón Lac , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutagénesis Insercional , Mutación , Especificidad de Órganos , Fenotipo , Embarazo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
In this study we extend the mouse Pax6 mutant allelic series to include a homozygous and hemizygous viable hypomorph allele. The Pax6(132-14Neu) allele is a Phe272Ile missense mutation within the third helix of the homeodomain. The mutant Pax6 homeodomain shows greatly reduced binding activity to the P3 DNA binding target. Glucagon-promoter activation by the entire mutant Pax6 product of a reporter gene driven by the G1 paired and homeodomain DNA binding target was slightly increased. We constructed mutant Pax6 genotypes such that Pax6 activity ranged between 100 and 0% and show that the extent of eye development is progressively reduced as Pax6 activity decreased. Two apparent thresholds identify three groups in which the extent of eye development abruptly shifted from complete eye at the highest levels of Pax6 to a rudimentary eye at intermediate levels of Pax6 to very early termination of eye development at the lowest levels of Pax6. Of the two Pax6-positive regions that participate in eye development, the surface ectoderm, which develops into the lens vesicle and the cornea, is more sensitive to reduced levels of Pax6 activity than the optic vesicle, which develops into the inner and outer retinal layers.
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Proteínas del Ojo/metabolismo , Ojo/embriología , Proteínas de Homeodominio/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Animales , Cruzamiento , Mapeo Cromosómico , ADN/metabolismo , Proteínas del Ojo/genética , Femenino , Fertilidad , Regulación del Desarrollo de la Expresión Génica , Genotipo , Glucagón/genética , Heterocigoto , Proteínas de Homeodominio/genética , Masculino , Ratones , Ratones Mutantes , Tamaño de los Órganos , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Fenotipo , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Represoras/genéticaRESUMEN
It is unclear what role vision plays in guiding mouse behaviour, since the mouse eye is of comparably low optical quality, and mice are considered to rely primarily on other senses. All C3H substrains are homozygous for the Pde6b(rd1) mutation and get blind by weaning age. To study the impact of the Pde6b(rd1) mutation on mouse behaviour and physiology, sighted C3H (C3H.Pde6b+) and normal C3H/HeH mice were phenotyped for different aspects. We confirmed retinal degeneration 1 in C3H/HeH mice, and the presence of a morphologically normal retina as well as visual ability in C3H.Pde6b+ mice. However, C3H.Pde6b+ mice showed an abnormal retinal function in the electroretinogram response, indicating that their vision was not normal as expected. C3H.Pde6b+ mice showed reduced latencies for several behaviours without any further alterations in these behaviours in comparison to C3H/HeH mice, suggesting that visual ability, although impaired, enables earlier usage of the behavioural repertoire in a novel environment, but does not lead to increased activity levels. These results emphasize the importance of comprehensive behavioural and physiological phenotyping.