42 results on '"Hamard, G."'
Search Results
2. Liver-targeted disruption of Apc in mice activates [beta]-catenin signaling and leads to hepatocellular carcinomas
- Author
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Colnot, S., Decaens, T., Niwa-Kawakita, M., Godard, C., Hamard, G., Kahn, A., Giovannini, M., and Perret, C.
- Subjects
Liver -- Research ,Science and technology - Abstract
Although inappropriate activation of the Wnt/[beta]-catenin pathway has been implicated in the development of hepatocellular carcinoma (HCC), the role of this signaling in liver carcinogenesis remains unclear. To investigate this issue, we constructed a mutant mouse strain, [Apc.sup.low/lox], in which exon 14 of the tumor-suppressor gene adenomatous polyposis coil (Apc) is flanked by loxP sequences, i.v. injection of adenovirus encoding Cre recombinase (AdCre) at high multiplicity [[10.sup.9] plaque-forming units (pfu) per mouse] inactivated the Apc gene in the liver and resulted in marked hepatomegaly, hepatocyte hyperplasia, and rapid mortality. [beta]-Catenin signaling activation was demonstrated by nuclear and cytoplasmic accumulation of [beta]-catenin in the hepatocytes and by the induction of [beta]-catenin target genes (glutamine synthetase, glutamate transporter 1, ornithine aminotransferase, and leukocyte cell-derived chemotaxin 2) in the liver. To test a long-term oncogenic effect, we inoculated mice with lower doses of AdCre (0.5 x [10.sup.9] pfu per mouse), compatible with both survival and persistence of [beta]-catenin-activated cells. In these conditions, 67% of mice developed HCC. [beta]-Catenin signaling was strongly activated in these Apc-inactivated HCCs. The HCCs were well, moderately, or poorly differentiated. Indeed, their histological and molecular features mimicked human HCC. Thus, deletion of Apc in the liver provides a valuable model of human HCC, and, in this model, activation of the Wnt/[beta]-catenin pathway by invalidation of Apc is required for liver tumorigenesis. adenomatous polyposis coil | hepatocarcinogenesis | mouse model
- Published
- 2004
3. AKR1B7 REGULATES ADIPOSE TISSUE EXPANSION VIA ITS PGF2A SYNTHASE ACTIVITY.: 415 accepted poster
- Author
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Pointud, J. C., Volat, F., Pastel, E., Morio, B., Sion, B., Hamard, G., Guichardant, M., Colas, R., Lefrançois-Martinez, A. M., and Martinez, A.
- Published
- 2012
4. De novo DNA microdeletion in a girl with Turner syndrome and Duchenne muscular dystrophy
- Author
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Chelly, J., Marlhens, F., Le Marec, B., Jeanpierre, M., Lambert, M., Hamard, G., Dutrillaux, B., and Kaplan, J. -C.
- Published
- 1986
- Full Text
- View/download PDF
5. Sumoylation by Ubc9 Regulates the Stem Cell Compartment and Structure and Function of the Intestinal Epithelium in Mice
- Author
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Demarque, M.D., Nacerddine, K., Neyret-Kahn, H., Andrieux, A., Danenberg, E.M., Jouvion, G., Bomme, P., Hamard, G., Romagnolo, B., Terris, B., Cumano, A., Barker, N., Clevers, H., Dejean, A., Demarque, M.D., Nacerddine, K., Neyret-Kahn, H., Andrieux, A., Danenberg, E.M., Jouvion, G., Bomme, P., Hamard, G., Romagnolo, B., Terris, B., Cumano, A., Barker, N., Clevers, H., and Dejean, A.
- Abstract
BACKGROUND & AIMS:: Small ubiquitin-like modifiers (SUMOs) are attached to other proteins to regulate their function (sumoylation). We investigated the role of Ubc9, which covalently attaches SUMOs to proteins, in the gastrointestinal tract of adult mice. METHODS:: We investigated the effects of decreased sumoylation in adult mammals by generating mice with an inducible knockout (by injection of 4-hydroxytamoxifen) of the E2 enzyme Ubc9 (Ubc9fl/-/ROSA26-CreERT2 mice). We analyzed the phenotypes using a range of histologic techniques. RESULTS:: Loss of Ubc9 from adult mice primarily affected the small intestine. Ubc9fl/-/ROSA26-CreERT2 mice died within 6 days of 4-hydroxytamoxifen injection, losing 20% or less of their body weight and developing severe diarrhea on the second day after injection. Surprisingly, other epithelial tissues appeared to be unaffected at that stage. Decreased sumoylation led to the depletion of the intestinal proliferative compartment and to the rapid disappearance of stem cells. Sumoylation was required to separate the proliferative and differentiated compartments from the crypt and control differentiation and function of the secretory lineage. Sumoylation was required for nucleus positioning and polarized organization of actin in the enterocytes. Loss of sumoylation caused detachment of the enterocytes from the basal lamina, as observed in tissue fragility diseases. We identified the intermediate filament keratin 8 as a SUMO substrate in epithelial cells. CONCLUSIONS:: Sumoylation maintains intestinal stem cells and the architecture, mechanical stability, and function of the intestinal epithelium of mice., BACKGROUND & AIMS:: Small ubiquitin-like modifiers (SUMOs) are attached to other proteins to regulate their function (sumoylation). We investigated the role of Ubc9, which covalently attaches SUMOs to proteins, in the gastrointestinal tract of adult mice. METHODS:: We investigated the effects of decreased sumoylation in adult mammals by generating mice with an inducible knockout (by injection of 4-hydroxytamoxifen) of the E2 enzyme Ubc9 (Ubc9fl/-/ROSA26-CreERT2 mice). We analyzed the phenotypes using a range of histologic techniques. RESULTS:: Loss of Ubc9 from adult mice primarily affected the small intestine. Ubc9fl/-/ROSA26-CreERT2 mice died within 6 days of 4-hydroxytamoxifen injection, losing 20% or less of their body weight and developing severe diarrhea on the second day after injection. Surprisingly, other epithelial tissues appeared to be unaffected at that stage. Decreased sumoylation led to the depletion of the intestinal proliferative compartment and to the rapid disappearance of stem cells. Sumoylation was required to separate the proliferative and differentiated compartments from the crypt and control differentiation and function of the secretory lineage. Sumoylation was required for nucleus positioning and polarized organization of actin in the enterocytes. Loss of sumoylation caused detachment of the enterocytes from the basal lamina, as observed in tissue fragility diseases. We identified the intermediate filament keratin 8 as a SUMO substrate in epithelial cells. CONCLUSIONS:: Sumoylation maintains intestinal stem cells and the architecture, mechanical stability, and function of the intestinal epithelium of mice.
- Published
- 2010
6. Hearing Is Normal without Connexin30
- Author
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Boulay, A.-C., primary, del Castillo, F. J., additional, Giraudet, F., additional, Hamard, G., additional, Giaume, C., additional, Petit, C., additional, Avan, P., additional, and Cohen-Salmon, M., additional
- Published
- 2013
- Full Text
- View/download PDF
7. A missense mutation in podocin leads to early and severe renal disease in mice
- Author
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Philippe, A., primary, Weber, S., additional, Esquivel, E.L., additional, Houbron, C., additional, Hamard, G., additional, Ratelade, J., additional, Kriz, W., additional, Schaefer, F., additional, Gubler, M.-C., additional, and Antignac, C., additional
- Published
- 2008
- Full Text
- View/download PDF
8. CO12 - Le remplacement du gène du récepteur AT1A de l’angiotensine II par un mutant « gain de fonction » produit un modèle original d’hypertension artérielle chez la souris
- Author
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Billet, S., primary, Hamard, G., additional, Bardin, S., additional, Baudrie, V., additional, Muffat-Joly, M., additional, Michaud, A., additional, Escoubet, B., additional, Elghozi, J.L., additional, Gasc, J.M., additional, Corvol, P., additional, and Clauser, E., additional
- Published
- 2005
- Full Text
- View/download PDF
9. Kinetics of atrazine degradation by photocatalytic process in aqueous solution
- Author
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Zahraa, O., primary, Sauvanaud, L., additional, Hamard, G., additional, and Bouchy, M., additional
- Published
- 2003
- Full Text
- View/download PDF
10. Depressed levels of prostaglandin F2α in mice lacking Akr1b7 increase basal adiposity and predispose to diet-induced obesity.
- Author
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Volat FE, Pointud JC, Pastel E, Morio B, Sion B, Hamard G, Guichardant M, Colas R, Lefrançois-Martinez AM, Martinez A, Volat, Fanny E, Pointud, Jean-Christophe, Pastel, Emilie, Morio, Béatrice, Sion, Benoit, Hamard, Ghislaine, Guichardant, Michel, Colas, Romain, Lefrançois-Martinez, Anne-Marie, and Martinez, Antoine
- Abstract
Negative regulators of white adipose tissue (WAT) expansion are poorly documented in vivo. Prostaglandin F(2α) (PGF(2α)) is a potent antiadipogenic factor in cultured preadipocytes, but evidence for its involvement in physiological context is lacking. We previously reported that Akr1b7, an aldo-keto reductase enriched in adipose stromal vascular fraction but absent from mature adipocytes, has antiadipogenic properties possibly supported by PGF(2α) synthase activity. To test whether lack of Akr1b7 could influence WAT homeostasis in vivo, we generated Akr1b7(-/-) mice in 129/Sv background. Akr1b7(-/-) mice displayed excessive basal adiposity resulting from adipocyte hyperplasia/hypertrophy and exhibited greater sensitivity to diet-induced obesity. Following adipose enlargement and irrespective of the diet, they developed liver steatosis and progressive insulin resistance. Akr1b7 loss was associated with decreased PGF(2α) WAT contents. Cloprostenol (PGF(2α) agonist) administration to Akr1b7(-/-) mice normalized WAT expansion by affecting both de novo adipocyte differentiation and size. Treatment of 3T3-L1 adipocytes and Akr1b7(-/-) mice with cloprostenol suggested that decreased adipocyte size resulted from inhibition of lipogenic gene expression. Hence, Akr1b7 is a major regulator of WAT development through at least two PGF(2α)-dependent mechanisms: inhibition of adipogenesis and lipogenesis. These findings provide molecular rationale to explore the status of aldo-keto reductases in dysregulations of adipose tissue homeostasis. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
11. Illegitimate transcription. Application to the analysis of truncated transcripts of the dystrophin gene in nonmuscle cultured cells from Duchenne and Becker patients.
- Author
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Chelly, J, primary, Gilgenkrantz, H, additional, Hugnot, J P, additional, Hamard, G, additional, Lambert, M, additional, Récan, D, additional, Akli, S, additional, Cometto, M, additional, Kahn, A, additional, and Kaplan, J C, additional
- Published
- 1991
- Full Text
- View/download PDF
12. Liver-targeted disruption of Apc in mice activates βcatenin signaling and leads to hepatocellular carcinomas.
- Author
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Colnot, S., Decaens, T., Niwa-Kawakita, M., Godard, C., Hamard, G., Kahn, A., Giovannini, M., and Perret, C.
- Subjects
BILIARY tract ,ABDOMEN ,LIVER cancer ,CARCINOGENESIS ,LIVER cells ,AMINO acids - Abstract
Although inappropriate activation of the Wnt/β-catenin pathway has been implicated in the development of hepatocellular carcinoma (HCC), the role of this signaling in liver carcinogenesis remains unclear. To investigate this issue, we constructed a mutant mouse strain, Apc
lox/lox , in which exon 14 of the tumor-suppressor gene adenomatous polyposis coli (Apc) is flanked by loxP sequences. i.v. injection of adenovirus encoding Cre recombinase (AdCre) at high multiplicity (109 plaque-forming units (pfu) per mouse) inactivated the Apc gene in the liver and resulted in marked hepatomegaly, hepatocyte hyperplasia, and rapid mortality. β-Catenin signaling activation was demonstrated by nuclear and cytoplasmic accumulation of β-catenin in the hepatocytes and by the induction of β-catenin target genes (glutamine synthetase, glutamate transporter 1, ornithine aminotransferase, and leukocyte cell-derived chemotaxin 2) in the liver. To test a long-term oncogenic effect, we inoculated mice with lower doses of AdCre (0.5 × 109 pfu per mouse), compatible with both survival and persistence of β-catenin-activated cells. In these conditions, 67% of mice developed HCC. β-Catenin signaling was strongly activated in these Apc-inactivated HCCs. The HCCs were well, moderately, or poorly differentiated. Indeed, their histological and molecular features mimicked human HCC. Thus, deletion of Apc in the liver provides a valuable model of human HCC, and, in this model, activation of the Wnt/β-catenin pathway by invalidation of Apc is required for liver tumorigenesis. [ABSTRACT FROM AUTHOR]- Published
- 2004
- Full Text
- View/download PDF
13. CONTRIBUTION A L'ÉTUDE DES PSOCOPTÈRES (II)APERÇU SYSTÉMATIQUE SUR LA FAUNE DE PSOCOPTERADE LA RÉGION DE RENNES (BRETAGNE, FRANCE)COMPRENANT NOTAMMENT DEUX ESPÈCES NOUVELLESPOUR LA FAUNE FRANÇAISEELIPSOCUS NUPTIALIS Roesler,ELIPSOCUS PALLIDUS Jentsh. (i)
- Author
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Hamard, G., Observatoire océanologique de Banyuls (OOB), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), and HAL UPMC, Gestionnaire
- Subjects
[SDV] Life Sciences [q-bio] ,[SDV]Life Sciences [q-bio] ,ComputingMilieux_MISCELLANEOUS - Abstract
International audience
- Published
- 1959
14. Dystrophin gene transcribed from different promoters in neuronal and glial cells.
- Author
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Chelly, J. and Hamard, G.
- Subjects
- *
BRAIN research - Abstract
Demonstrates that the brain-type promoter of the dystrophin gene is highly specific to neurons, in which there is a significant increase in the amount of brain-specific messenger RNA during the course of `in vitro' maturation. Methods; Results; Discussion.
- Published
- 1990
- Full Text
- View/download PDF
15. Cre-mediated germline mosaicism: a method allowing rapid generation of several alleles of a target gene.
- Author
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Holzenberger, M, Lenzner, C, Leneuve, P, Zaoui, R, Hamard, G, Vaulont, S, and Bouc, Y L
- Abstract
Conditional gene targeting uses the insertion of expression cassettes for the selection of targeted embryonic stem cells. The presence of these cassettes in the final targeted chromosomal locus may affect the normal expression of the targeted gene and produce interesting knock down phenotypes. We show here that the selection cassette may then be selectively removed in vivo, using three appropriately positioned loxP sites in the targeted gene and the transgenic mouse EIIaCre. This strategy was applied to two different target genes and we demonstrated that it is reliable and reproducible. First, we generated double transgenic EIIaCre/loxP mice (F1) that showed variable degrees of mosaicism for partially CRE-recombined floxed alleles. Efficiency of EIIaCre at creating mosaicism was dependent on the target gene and on parental transmission of the transgene. The segregation of partially recombined alleles and EIIaCre transgene was obtained in the next generation using mosaic F1 males. Mosaic females were unsuitable for this purpose because they systematically generated complete excisions during oogenesis. Our strategy is applicable to other approaches based on three loxP sites. As this procedure allows generation of knock down (presence of neo), knockout (total exision of the loxP-flanked sequences) and floxed substrains (excision of the selection cassette) from a single, targeted germline mutation and in a single experiment, its use may become more widespread in conditional mutagenesis.
- Published
- 2000
- Full Text
- View/download PDF
16. Cre-mediated germline mosaicism: a method allowing rapid generation of several alleles of a target gene
- Author
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Lenzner, C., Hamard, G., Vaulont, S., Holzenberger, M., Leneuve, P., Zaoui, R., and Bouc, Y.L.
- Abstract
Conditional gene targeting uses the insertion of expression cassettes for the selection of targeted embryonic stem cells. The presence of these cassettes in the final targeted chromosomal locus may affect the normal expression of the targeted gene and produce interesting knock down phenotypes. We show here that the selection cassette may then be selectively removed in vivo, using three appropriately positioned loxP sites in the targeted gene and the transgenic mouse EIIaCre. This strategy was applied to two different target genes and we demonstrated that it is reliable and reproducible. First, we generated double transgenic EIIaCre/loxP mice (F1) that showed variable degrees of mosaicism for partially Cre-recombined floxed alleles. Efficiency of EIIaCre at creating mosaicism was dependent on the target gene and on parental transmission of the transgene. The segregation of partially recombined alleles and EIIaCre transgene was obtained in the next generation using mosaic F1 males. Mosaic females were unsuitable for this purpose because they systematically generated complete excisions during oogenesis. Our strategy is applicable to other approaches based on three loxP sites. As this procedure allows generation of knock down (presence of neo), knockout (total exision of the loxP-flanked sequences) and floxed substrains (excision of the selection cassette) from a single, targeted germline mutation and in a single experiment, its use may become more widespread in conditional mutagenesis.
- Published
- 2000
17. The AMPKγ1 subunit plays an essential role in erythrocyte membrane elasticity, and its genetic inactivation induces splenomegaly and anemia.
- Author
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Foretz M, Hébrard S, Guihard S, Leclerc J, Do Cruzeiro M, Hamard G, Niedergang F, Gaudry M, and Viollet B
- Subjects
- AMP-Activated Protein Kinases genetics, Anemia blood, Anemia genetics, Anemia, Hemolytic enzymology, Anemia, Hemolytic genetics, Animals, Blotting, Western, Elasticity, Erythroblasts metabolism, Erythroblasts pathology, Erythrocyte Count, Erythrocyte Deformability, Female, Hyperplasia, Iron metabolism, Macrophages metabolism, Macrophages pathology, Male, Membrane Proteins metabolism, Mice, Mice, 129 Strain, Mice, Inbred C57BL, Mice, Knockout, Spleen metabolism, Spleen pathology, Splenomegaly blood, Splenomegaly genetics, AMP-Activated Protein Kinases metabolism, Anemia enzymology, Erythrocyte Membrane metabolism, Splenomegaly enzymology
- Abstract
AMP-activated protein kinase (AMPK) is an αβγ heterotrimer conserved throughout evolution and important for energy sensing in all eukaryote cells. AMPK controls metabolism and various cellular events in response to both hormones and changes in cellular energy status. The γ subunit senses intracellular energy status through the competitive binding of AMP and ATP. We show here that targeted disruption of the mouse AMPKγ1 gene (Prkag1) causes regenerative hemolytic anemia by increasing the sequestration of abnormal erythrocytes. Prkag1(-/-) mice displayed splenomegaly and iron accumulation due to compensatory splenic erythropoiesis and erythrophagocytosis. Moreover, AMPKγ1-deficient erythrocytes were highly resistant to osmotic hemolysis and poorly deformable in response to increasing shear stress, consistent with greater membrane rigidity. No change in cytoskeletal protein composition was observed; however, the phosphorylation level of adducin, a protein promoting the binding of spectrin to actin, was higher in AMPKγ1-deficient erythrocytes. Together, these results demonstrate that AMPKγ1 subunit is required for the maintenance of erythrocyte membrane elasticity.
- Published
- 2011
- Full Text
- View/download PDF
18. Sumoylation by Ubc9 regulates the stem cell compartment and structure and function of the intestinal epithelium in mice.
- Author
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Demarque MD, Nacerddine K, Neyret-Kahn H, Andrieux A, Danenberg E, Jouvion G, Bomme P, Hamard G, Romagnolo B, Terris B, Cumano A, Barker N, Clevers H, and Dejean A
- Subjects
- Animals, Intestinal Mucosa drug effects, Intestinal Mucosa pathology, Mice, Mice, Knockout, Phenotype, Stem Cells drug effects, Tamoxifen analogs & derivatives, Tamoxifen pharmacology, Ubiquitin-Conjugating Enzymes genetics, Intestinal Mucosa metabolism, Stem Cells metabolism, Sumoylation, Ubiquitin-Conjugating Enzymes metabolism
- Abstract
Background & Aims: Small ubiquitin-like modifiers (SUMOs) are attached to other proteins to regulate their function (sumoylation). We investigated the role of Ubc9, which covalently attaches SUMOs to proteins, in the gastrointestinal tract of adult mice., Methods: We investigated the effects of decreased sumoylation in adult mammals by generating mice with an inducible knockout (by injection of 4-hydroxytamoxifen) of the E2 enzyme Ubc9 (Ubc9fl/-/ROSA26-CreERT2 mice). We analyzed the phenotypes using a range of histologic techniques., Results: Loss of Ubc9 from adult mice primarily affected the small intestine. Ubc9fl/-/ROSA26-CreERT2 mice died within 6 days of 4-hydroxytamoxifen injection, losing 20% or less of their body weight and developing severe diarrhea on the second day after injection. Surprisingly, other epithelial tissues appeared to be unaffected at that stage. Decreased sumoylation led to the depletion of the intestinal proliferative compartment and to the rapid disappearance of stem cells. Sumoylation was required to separate the proliferative and differentiated compartments from the crypt and control differentiation and function of the secretory lineage. Sumoylation was required for nucleus positioning and polarized organization of actin in the enterocytes. Loss of sumoylation caused detachment of the enterocytes from the basal lamina, as observed in tissue fragility diseases. We identified the intermediate filament keratin 8 as a SUMO substrate in epithelial cells., Conclusions: Sumoylation maintains intestinal stem cells and the architecture, mechanical stability, and function of the intestinal epithelium of mice., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
19. Cubilin is essential for albumin reabsorption in the renal proximal tubule.
- Author
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Amsellem S, Gburek J, Hamard G, Nielsen R, Willnow TE, Devuyst O, Nexo E, Verroust PJ, Christensen EI, and Kozyraki R
- Subjects
- Absorption, Animals, DNA-Binding Proteins metabolism, Disease Models, Animal, Integrases genetics, Low Density Lipoprotein Receptor-Related Protein-2 genetics, Low Density Lipoprotein Receptor-Related Protein-2 metabolism, Mice, Mice, Inbred C57BL, Mice, Knockout, Receptors, Cell Surface genetics, Transcription Factors metabolism, Vitamin B 12 metabolism, Albumins metabolism, Kidney Tubules, Proximal metabolism, Proteinuria metabolism, Receptors, Cell Surface metabolism
- Abstract
Receptor-mediated endocytosis is responsible for protein reabsorption in the proximal tubule. This process involves two interacting receptors, megalin and cubilin, which form a complex with amnionless. Whether these proteins function in parallel or as part of an integrated system is not well understood. Here, we report the renal effects of genetic ablation of cubilin, with or without concomitant ablation of megalin, using a conditional Cre-loxP system. We observed that proximal tubule cells did not localize amnionless to the plasma membrane in the absence of cubilin, indicating a mutual dependency of cubilin and amnionless to form a functional membrane receptor complex. The cubilin-amnionless complex mediated internalization of intrinsic factor-vitamin B12 complexes, but megalin considerably increased the uptake. Furthermore, cubilin-deficient mice exhibited markedly decreased uptake of albumin by proximal tubule cells and resultant albuminuria. Inactivation of both megalin and cubilin did not increase albuminuria, indicating that the main role of megalin in albumin reabsorption is to drive the internalization of cubilin-albumin complexes. In contrast, cubulin deficiency did not affect urinary tubular uptake or excretion of vitamin D-binding protein (DBP), which binds cubilin and megalin. In addition, we observed cubilin-independent reabsorption of the "specific" cubilin ligands transferrin, CC16, and apoA-I, suggesting a role for megalin and perhaps other receptors in their reabsorption. In summary, with regard to albumin, cubilin is essential for its reabsorption by proximal tubule cells, and megalin drives internalization of cubilin-albumin complexes. These genetic models will allow further analysis of protein trafficking in the progression of proteinuric renal diseases.
- Published
- 2010
- Full Text
- View/download PDF
20. A murine model of Denys-Drash syndrome reveals novel transcriptional targets of WT1 in podocytes.
- Author
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Ratelade J, Arrondel C, Hamard G, Garbay S, Harvey S, Biebuyck N, Schulz H, Hastie N, Pontoglio M, Gubler MC, Antignac C, and Heidet L
- Subjects
- Alleles, Animals, Cell Line, Cytochrome P-450 Enzyme System metabolism, Denys-Drash Syndrome enzymology, Disease Models, Animal, Gene Expression Profiling, Gene Expression Regulation, Heterozygote, Kidney Glomerulus enzymology, Kidney Glomerulus pathology, Mesonephros enzymology, Mice, Mice, Inbred Strains, Mutation genetics, Protein Binding, Protein Transport, RNA Transport, Regulatory Sequences, Nucleic Acid genetics, Retinoic Acid 4-Hydroxylase, Sulfotransferases genetics, Denys-Drash Syndrome genetics, Podocytes metabolism, Transcription, Genetic, WT1 Proteins metabolism
- Abstract
The Wilms tumor-suppressor gene WT1, a key player in renal development, also has a crucial role in maintenance of the glomerulus in the mature kidney. However, molecular pathways orchestrated by WT1 in podocytes, where it is highly expressed, remain unknown. Their defects are thought to modify the cross-talk between podocytes and other glomerular cells and ultimately lead to glomerular sclerosis, as observed in diffuse mesangial sclerosis (DMS) a nephropathy associated with WT1 mutations. To identify podocyte WT1 targets, we generated a novel DMS mouse line, performed gene expression profiling in isolated glomeruli and identified excellent candidates that may modify podocyte differentiation and growth factor signaling in glomeruli. Scel, encoding sciellin, a protein of the cornified envelope in the skin, and Sulf1, encoding a 6-O endosulfatase, are shown to be expressed in wild-type podocytes and to be strongly down-regulated in mutants. Co-expression of Wt1, Scel and Sulf1 was also found in a mesonephric cell line, and siRNA-mediated knockdown of WT1 decreased Scel and Sulf1 mRNAs and proteins. By ChIP we show that Scel and Sulf1 are direct WT1 targets. Cyp26a1, encoding an enzyme involved in the degradation of retinoic acid, is shown to be up-regulated in mutant podocytes. Cyp26a1 may play a role in the development of glomerular lesions but does not seem to be regulated by WT1. These results provide novel clues in our understanding of normal glomerular function and early events involved in glomerulosclerosis.
- Published
- 2010
- Full Text
- View/download PDF
21. Stereocilin-deficient mice reveal the origin of cochlear waveform distortions.
- Author
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Verpy E, Weil D, Leibovici M, Goodyear RJ, Hamard G, Houdon C, Lefèvre GM, Hardelin JP, Richardson GP, Avan P, and Petit C
- Subjects
- Acoustic Stimulation, Animals, Female, Gene Expression Regulation, Hair Cells, Auditory cytology, Hair Cells, Auditory ultrastructure, Immunohistochemistry, Intercellular Signaling Peptides and Proteins, Male, Mice, Mice, Knockout, Cochlea physiology, Hair Cells, Auditory metabolism, Proteins genetics, Proteins metabolism
- Abstract
Although the cochlea is an amplifier and a remarkably sensitive and finely tuned detector of sounds, it also produces conspicuous mechanical and electrical waveform distortions. These distortions reflect nonlinear mechanical interactions within the cochlea. By allowing one tone to suppress another (masking effect), they contribute to speech intelligibility. Tones can also combine to produce sounds with frequencies not present in the acoustic stimulus. These sounds compose the otoacoustic emissions that are extensively used to screen hearing in newborns. Because both cochlear amplification and distortion originate from the outer hair cells-one of the two types of sensory receptor cells-it has been speculated that they stem from a common mechanism. Here we show that the nonlinearity underlying cochlear waveform distortions relies on the presence of stereocilin, a protein defective in a recessive form of human deafness. Stereocilin was detected in association with horizontal top connectors, lateral links that join adjacent stereocilia within the outer hair cell's hair bundle. These links were absent in stereocilin-null mutant mice, which became progressively deaf. At the onset of hearing, however, their cochlear sensitivity and frequency tuning were almost normal, although masking was much reduced and both acoustic and electrical waveform distortions were completely lacking. From this unique functional situation, we conclude that the main source of cochlear waveform distortions is a deflection-dependent hair bundle stiffness resulting from constraints imposed by the horizontal top connectors, and not from the intrinsic nonlinear behaviour of the mechanoelectrical transducer channel.
- Published
- 2008
- Full Text
- View/download PDF
22. Gain-of-function mutant of angiotensin II receptor, type 1A, causes hypertension and cardiovascular fibrosis in mice.
- Author
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Billet S, Bardin S, Verp S, Baudrie V, Michaud A, Conchon S, Muffat-Joly M, Escoubet B, Souil E, Hamard G, Bernstein KE, Gasc JM, Elghozi JL, Corvol P, and Clauser E
- Subjects
- Angiotensins metabolism, Animals, Asparagine genetics, Asparagine metabolism, Blood Pressure, Cardiovascular Diseases genetics, Cardiovascular Diseases physiopathology, Disease Progression, Female, Fibrosis metabolism, Fibrosis pathology, Gene Expression Regulation, Hyperaldosteronism complications, Hyperaldosteronism metabolism, Hyperaldosteronism pathology, Hypertension genetics, Hypertension physiopathology, Kidney physiology, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Mutation genetics, Receptor, Angiotensin, Type 1 genetics, Renin blood, Signal Transduction, Cardiovascular Diseases metabolism, Cardiovascular Diseases pathology, Hypertension metabolism, Hypertension mortality, Receptor, Angiotensin, Type 1 metabolism
- Abstract
The role of the renin-angiotensin system has been investigated by overexpression or inactivation of its different genes in animals. However, there is no data concerning the effect of the constitutive activation of any component of the system. A knockin mouse model has been constructed with a gain-of-function mutant of the Ang II receptor, type 1A (AT(1A)), associating a constitutively activating mutation (N111S) with a C-terminal deletion, which impairs receptor internalization and desensitization. In vivo consequences of this mutant receptor expression in homozygous mice recapitulate its in vitro characteristics: the pressor response is more sensitive to Ang II and longer lasting. These mice present with a moderate (~20 mmHg) and stable increase in BP. They also develop early and progressive renal fibrosis and cardiac fibrosis and diastolic dysfunction. However, there was no overt cardiac hypertrophy. The hormonal parameters (low-renin and inappropriately normal aldosterone productions) mimic those of low-renin human hypertension. This new model reveals that a constitutive activation of AT(1A) leads to cardiac and renal fibrosis in spite of a modest effect on BP and will be useful for investigating the role of Ang II in target organs in a model similar to some forms of human hypertension.
- Published
- 2007
- Full Text
- View/download PDF
23. Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse.
- Author
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Roux I, Safieddine S, Nouvian R, Grati M, Simmler MC, Bahloul A, Perfettini I, Le Gall M, Rostaing P, Hamard G, Triller A, Avan P, Moser T, and Petit C
- Subjects
- Animals, Auditory Pathways metabolism, Calcium metabolism, Cochlea growth & development, Deafness genetics, Deafness physiopathology, Evoked Potentials, Auditory, Brain Stem, Hair Cells, Auditory, Inner ultrastructure, Humans, Membrane Fusion, Membrane Proteins deficiency, Membrane Proteins genetics, Mice, Mice, Inbred C57BL, Mice, Knockout, Synaptic Transmission, Synaptic Vesicles metabolism, Synaptosomal-Associated Protein 25 metabolism, Syntaxin 1 metabolism, Time Factors, Cochlea metabolism, Deafness metabolism, Exocytosis, Hair Cells, Auditory, Inner metabolism, Membrane Proteins metabolism, Synapses metabolism
- Abstract
The auditory inner hair cell (IHC) ribbon synapse operates with an exceptional temporal precision and maintains a high level of neurotransmitter release. However, the molecular mechanisms underlying IHC synaptic exocytosis are largely unknown. We studied otoferlin, a predicted C2-domain transmembrane protein, which is defective in a recessive form of human deafness. We show that otoferlin expression in the hair cells correlates with afferent synaptogenesis and find that otoferlin localizes to ribbon-associated synaptic vesicles. Otoferlin binds Ca(2+) and displays Ca(2+)-dependent interactions with the SNARE proteins syntaxin1 and SNAP25. Otoferlin deficient mice (Otof(-/-)) are profoundly deaf. Exocytosis in Otof(-/-) IHCs is almost completely abolished, despite normal ribbon synapse morphogenesis and Ca(2+) current. Thus, otoferlin is essential for a late step of synaptic vesicle exocytosis and may act as the major Ca(2+) sensor triggering membrane fusion at the IHC ribbon synapse.
- Published
- 2006
- Full Text
- View/download PDF
24. Targeted disruption of the hepcidin 1 gene results in severe hemochromatosis.
- Author
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Lesbordes-Brion JC, Viatte L, Bennoun M, Lou DQ, Ramey G, Houbron C, Hamard G, Kahn A, and Vaulont S
- Subjects
- Animals, Antimicrobial Cationic Peptides metabolism, Ferritins metabolism, Hemochromatosis metabolism, Hemochromatosis pathology, Hepcidins, Iron metabolism, Macrophages metabolism, Macrophages pathology, Mice, Mice, Knockout, Spleen metabolism, Spleen pathology, Upstream Stimulatory Factors deficiency, Upstream Stimulatory Factors metabolism, Antimicrobial Cationic Peptides deficiency, Gene Deletion, Hemochromatosis genetics, Open Reading Frames genetics, Quantitative Trait Loci genetics
- Abstract
We previously reported that mice made deficient for the transcriptional factor USF2 fail to express hepcidin 1 and hepcidin 2 genes as a consequence of targeted disruption of the Usf2 gene lying just upstream in the locus. These mice developed an iron overload phenotype with excess iron deposition in parenchymal cells and decreased reticuloendothelial iron. At that time, although the role of USF2 was still confounding, we proposed for the first time the role of hepcidin as a negative regulator of iron absorption and iron release from macrophages. Accordingly, we subsequently demonstrated that hyperexpression of hepcidin 1, but not hepcidin 2, resulted in a profound hyposideremic anemia. To analyze the consequences of hepcidin 1 deletion on iron metabolism without any disturbance due to USF2 deficiency, we disrupted the hepcidin 1 gene by targeting almost all the coding region. Confirming our prior results, Hepc1(-/-) mice developed early and severe multivisceral iron overload, with sparing of the spleen macrophages, and demonstrated increased serum iron and ferritin levels as compared with their controls.
- Published
- 2006
- Full Text
- View/download PDF
25. Branching and nucleokinesis defects in migrating interneurons derived from doublecortin knockout mice.
- Author
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Kappeler C, Saillour Y, Baudoin JP, Tuy FP, Alvarez C, Houbron C, Gaspar P, Hamard G, Chelly J, Métin C, and Francis F
- Subjects
- Animals, Cells, Cultured, Coculture Techniques, Doublecortin Domain Proteins, Doublecortin Protein, Female, Male, Median Eminence cytology, Median Eminence pathology, Mice, Mice, Inbred C57BL, Mice, Knockout, Microtubule-Associated Proteins physiology, Neuropeptides physiology, Organ Culture Techniques, Cell Movement genetics, Cell Nucleus genetics, Cell Nucleus metabolism, Interneurons pathology, Microtubule-Associated Proteins deficiency, Microtubule-Associated Proteins genetics, Neuropeptides deficiency, Neuropeptides genetics
- Abstract
Type I lissencephaly results from mutations in the doublecortin (DCX) and LIS1 genes. We generated Dcx knockout mice to further understand the pathophysiological mechanisms associated with this cortical malformation. Dcx is expressed in migrating interneurons in developing human and mouse brains. Video microscopy analyses of such tangentially migrating neuron populations derived from the medial ganglionic eminence show defects in migratory dynamics. Specifically, the formation and division of growth cones, leading to the production of new branches, are more frequent in knockout cells, although branches are less stable. Dcx-deficient cells thus migrate in a disorganized manner, extending and retracting short branches and making less long-distant movements of the nucleus. Despite these differences, migratory speeds and distances remain similar to wild-type cells. These novel data thus highlight a role for Dcx, a microtubule-associated protein enriched at the leading edge in the branching and nucleokinesis of migrating interneurons.
- Published
- 2006
- Full Text
- View/download PDF
26. Six1 and Six4 homeoproteins are required for Pax3 and Mrf expression during myogenesis in the mouse embryo.
- Author
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Grifone R, Demignon J, Houbron C, Souil E, Niro C, Seller MJ, Hamard G, and Maire P
- Subjects
- Animals, Apoptosis physiology, Bone and Bones abnormalities, DNA-Binding Proteins metabolism, Homeodomain Proteins genetics, Mice, Muscle Proteins metabolism, Muscles abnormalities, Muscles embryology, Myogenic Regulatory Factor 5, Myogenic Regulatory Factors metabolism, Myogenin, PAX3 Transcription Factor, Paired Box Transcription Factors, Trans-Activators deficiency, Transcription Factors metabolism, DNA-Binding Proteins genetics, Homeodomain Proteins metabolism, Muscle Development physiology, Muscle Proteins genetics, Myogenic Regulatory Factors genetics, Trans-Activators genetics, Trans-Activators metabolism, Transcription Factors genetics
- Abstract
In mammals, Six5, Six4 and Six1 genes are co-expressed during mouse myogenesis. Six4 and Six5 single knockout (KO) mice have no developmental defects, while Six1 KO mice die at birth and show multiple organ developmental defects. We have generated Six1Six4 double KO mice and show an aggravation of the phenotype previously reported for the single Six1 KO. Six1Six4 double KO mice are characterized by severe craniofacial and rib defects, and general muscle hypoplasia. At the limb bud level, Six1 and Six4 homeogenes control early steps of myogenic cell delamination and migration from the somite through the control of Pax3 gene expression. Impaired in their migratory pathway, cells of the somitic ventrolateral dermomyotome are rerouted, lose their identity and die by apoptosis. At the interlimb level, epaxial Met expression is abolished, while it is preserved in Pax3-deficient embryos. Within the myotome, absence of Six1 and Six4 impairs the expression of the myogenic regulatory factors myogenin and Myod1, and Mrf4 expression becomes undetectable. Myf5 expression is correctly initiated but becomes restricted to the caudal region of each somite. Early syndetomal expression of scleraxis is reduced in the Six1Six4 embryo, while the myotomal expression of Fgfr4 and Fgf8 but not Fgf4 and Fgf6 is maintained. These results highlight the different roles played by Six proteins during skeletal myogenesis.
- Published
- 2005
- Full Text
- View/download PDF
27. Lactadherin promotes VEGF-dependent neovascularization.
- Author
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Silvestre JS, Théry C, Hamard G, Boddaert J, Aguilar B, Delcayre A, Houbron C, Tamarat R, Blanc-Brude O, Heeneman S, Clergue M, Duriez M, Merval R, Lévy B, Tedgui A, Amigorena S, and Mallat Z
- Subjects
- Analysis of Variance, Animals, Blotting, Southern, Crosses, Genetic, Female, Genetic Vectors, Humans, Integrin alphaVbeta3 metabolism, Ischemia metabolism, Mice, Mice, Inbred C57BL, Muscle, Skeletal metabolism, Phosphorylation, Protein Serine-Threonine Kinases metabolism, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-akt, Angiogenesis Inducing Agents metabolism, Antigens, Surface metabolism, Milk Proteins metabolism, Neovascularization, Physiologic physiology, Signal Transduction physiology, Vascular Endothelial Growth Factor A metabolism
- Abstract
Vascular endothelial growth factor (VEGF)-induced blood vessel growth is involved in both physiological and pathological angiogenesis and requires integrin-mediated signaling. We now show that an integrin-binding protein initially described in milk-fat globule, MFG-E8 (also known as lactadherin), is expressed in and around blood vessels and has a crucial role in VEGF-dependent neovascularization in the adult mouse. Using neutralizing antibodies and lactadherin-deficient animals, we show that lactadherin interacts with alphavbeta3 and alphavbeta5 integrins and alters both VEGF-dependent Akt phosphorylation and neovascularization. In the absence of VEGF, lactadherin administration induced alphavbeta3- and alphavbeta5-dependent Akt phosphorylation in endothelial cells in vitro and strongly improved postischemic neovascularization in vivo. These results show a crucial role for lactadherin in VEGF-dependent neovascularization and identify lactadherin as an important target for the modulation of neovascularization.
- Published
- 2005
- Full Text
- View/download PDF
28. Deafness and cochlear fibrocyte alterations in mice deficient for the inner ear protein otospiralin.
- Author
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Delprat B, Ruel J, Guitton MJ, Hamard G, Lenoir M, Pujol R, Puel JL, Brabet P, and Hamel CP
- Subjects
- Animals, Auditory Threshold, Cochlea pathology, Cochlea physiopathology, Deafness genetics, Deafness pathology, Electrophysiology, Gene Targeting, Humans, Mice, Mice, Knockout, Proteins genetics, Cochlea cytology, Cochlea physiology, Deafness physiopathology, Proteins metabolism
- Abstract
In the cochlea, the mammalian auditory organ, fibrocytes of the mesenchymal nonsensory regions play important roles in cochlear physiology, including the maintenance of ionic and hydric components in the endolymph. Occurrence of human deafness in fibrocyte alterations underlines their critical roles in auditory function. We recently described a novel gene, Otos, which encodes otospiralin, a small protein of unknown function that is produced by the fibrocytes of the cochlea and vestibule. We now have generated mice with deletion of Otos and found that they show moderate deafness, with no frequency predominance. Histopathology revealed a degeneration of type II and IV fibrocytes, while hair cells and stria vascularis appeared normal. Together, these findings suggest that impairment of fibrocytes caused by the loss in otospiralin leads to abnormal cochlear physiology and auditory function. This moderate dysfunction may predispose to age-related hearing loss.
- Published
- 2005
- Full Text
- View/download PDF
29. Colorectal cancers in a new mouse model of familial adenomatous polyposis: influence of genetic and environmental modifiers.
- Author
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Colnot S, Niwa-Kawakita M, Hamard G, Godard C, Le Plenier S, Houbron C, Romagnolo B, Berrebi D, Giovannini M, and Perret C
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Clone Cells, Colon pathology, Colonic Neoplasms genetics, Colonic Neoplasms pathology, Colorectal Neoplasms etiology, Colorectal Neoplasms pathology, DNA, Neoplasm genetics, Disease Models, Animal, Environment, Exons, Gene Library, Mice, Mice, Knockout, Molecular Sequence Data, RNA, Neoplasm genetics, Rectal Neoplasms genetics, Rectal Neoplasms pathology, Sequence Deletion, Colorectal Neoplasms genetics, Colorectal Neoplasms physiopathology, Gene Deletion, Genes, APC physiology
- Abstract
Murine models of familial adenomatous polyposis harbor a germinal heterozygous mutation on Apc tumor suppressor gene. They are valuable tools for studying intestinal carcinogenesis, as most human sporadic cancers contain inactivating mutations of APC. However, Apc(+/-) mice, such as the well-characterized Apc(Min/+) model, develop cancers principally in the small intestine, while humans develop mainly colorectal cancers. We used a Cre-loxP strategy to achieve a new model of germline Apc invalidation in which exon 14 is deleted. We compared the phenotype of these Apc(Delta14/+) mice to that of the classical Apc(Min/+). The main phenotypic difference is the shift of the tumors in the distal colon and rectum, often associated with a rectal prolapse. Thus, the severity of the colorectal phenotype is partly due to the particular mutation Delta14, but also to environmental parameters, as mice raised in conventional conditions developed more colon cancers than those raised in pathogen-free conditions. All lesions, including early lesions, revealed Apc LOH and loss of Apc gene expression. They accumulated beta-catenin, overexpressed the beta-catenin target genes cyclin D1 and c-Myc, and the distribution pattern of glutamine synthetase, a beta-catenin target gene recently identified in the liver, was mosaic in intestinal adenomas. The Apc(Delta14/+) model is thus a useful new tool for studies on the molecular mechanisms of colorectal tumorigenesis.
- Published
- 2004
- Full Text
- View/download PDF
30. Targeted inactivation of serum response factor in the developing heart results in myocardial defects and embryonic lethality.
- Author
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Parlakian A, Tuil D, Hamard G, Tavernier G, Hentzen D, Concordet JP, Paulin D, Li Z, and Daegelen D
- Subjects
- Animals, Apoptosis, Base Sequence, Cell Division, DNA, Complementary genetics, Female, Fetal Death, Fetal Heart cytology, Fetal Heart metabolism, Gene Expression Regulation, Developmental, Gene Targeting, Gestational Age, Heart Defects, Congenital embryology, Heart Defects, Congenital etiology, Heart Defects, Congenital genetics, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, Mice, Transgenic, Organ Specificity, Pregnancy, Serum Response Factor antagonists & inhibitors, Serum Response Factor deficiency, Serum Response Factor physiology, Transcription Factors genetics, Fetal Heart embryology, Serum Response Factor genetics
- Abstract
Serum response factor (SRF) is at the confluence of multiple signaling pathways controlling the transcription of immediate-early response genes and muscle-specific genes. There are active SRF target sequences in more than 50 genes expressed in the three muscle lineages including normal and diseased hearts. However, the role of SRF in heart formation has not been addressed in vivo thus far due to the early requirement of SRF for mesoderm formation. We have generated a conditional mutant of SRF by using Cre-LoxP strategy that will be extremely useful to study the role of SRF in embryonic and postnatal cardiac functions, as well as in other tissues. This report shows that heart-specific deletion of SRF in the embryo by using a new beta MHC-Cre transgenic mouse line results in lethal cardiac defects between embryonic day 10.5 (E10.5) and E13.5, as evidenced by abnormally thin myocardium, dilated cardiac chambers, poor trabeculation, and a disorganized interventricular septum. At E9.5, we found a marked reduction in the expression of essential regulators of heart development, including Nkx2.5, GATA4, myocardin, and the SRF target gene c-fos prior to overt maldevelopment. We conclude that SRF is crucial for cardiac differentiation and maturation, acting as a global regulator of multiple developmental genes.
- Published
- 2004
- Full Text
- View/download PDF
31. Altered myogenesis in Six1-deficient mice.
- Author
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Laclef C, Hamard G, Demignon J, Souil E, Houbron C, and Maire P
- Subjects
- Animals, Apoptosis physiology, Cell Differentiation physiology, Cell Movement physiology, Embryo, Mammalian anatomy & histology, Embryo, Mammalian physiology, Female, Gene Targeting, Homeodomain Proteins genetics, In Situ Hybridization, In Situ Nick-End Labeling, Male, Mice, Mice, Transgenic, Muscle Development genetics, Muscle, Skeletal pathology, Muscle, Skeletal physiology, MyoD Protein genetics, MyoD Protein metabolism, Myogenin genetics, Myogenin metabolism, Phenotype, Ribs pathology, Sternum pathology, Homeodomain Proteins metabolism, Muscle Development physiology
- Abstract
Six homeoproteins are expressed in several tissues, including muscle, during vertebrate embryogenesis, suggesting that they may be involved in diverse differentiation processes. To determine the functions of the Six1 gene during myogenesis, we constructed Six1-deficient mice by replacing its first exon with the lacZ gene. Mice lacking Six1 die at birth because of severe rib malformations and show extensive muscle hypoplasia affecting most of the body muscles in particular certain hypaxial muscles. Six1(-/-) embryos have impaired primary myogenesis, characterized, at E13.5, by a severe reduction and disorganisation of primary myofibers in most body muscles. While Myf5, MyoD and myogenin are correctly expressed in the somitic compartment in early Six1(-/-) embryos, by E11.5 MyoD and myogenin gene activation is reduced and delayed in limb buds. However, this is not the consequence of a reduced ability of myogenic precursor cells to migrate into the limb buds or of an abnormal apoptosis of myoblasts lacking Six1. It appears therefore that Six1 plays a specific role in hypaxial muscle differentiation, distinct from those of other hypaxial determinants such as Pax3, cMet, Lbx1 or Mox2.
- Published
- 2003
- Full Text
- View/download PDF
32. Cre-mediated germline mosaicism: a new transgenic mouse for the selective removal of residual markers from tri-lox conditional alleles.
- Author
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Leneuve P, Colnot S, Hamard G, Francis F, Niwa-Kawakita M, Giovannini M, and Holzenberger M
- Subjects
- Alleles, Animals, Animals, Newborn, Female, Gene Deletion, Integrases genetics, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Recombination, Genetic genetics, Viral Proteins genetics, Germ-Line Mutation, Integrases metabolism, Mosaicism genetics, Viral Proteins metabolism
- Abstract
The binary Cre-lox conditional knockout system requires an essential part of the target gene to be flanked by loxP sites, enabling excision in vivo upon Cre expression. LoxP sites are introduced by homologous recombination, together with a selectable marker. However, this marker can disturb gene expression and should be removed. The marker is therefore often prepared with a third, flanking loxP site (tri-lox construct), facilitating its selective removal by partial Cre-lox recombination. We have shown that this excision can be achieved in vivo in the germline using EIIaCre transgenic mice, and have described the advantages of in vivo over in vitro removal. We show here that MeuCre40, a new transgenic mouse, more reliably and reproducibly generates an optimal partial mosaic Cre-lox recombination pattern in the early embryo. This mosaicism was transmitted to the germline and to many other tissues. Alleles with partial deletions, in particular floxed alleles from which the selectable marker was removed, were readily recovered in the next generation, after segregation from the transgene. Segregation via paternal or maternal transmission led to successful recovery of the alleles of interest. We also obtained total deletion of the floxed regions in the same experiment, making this transgene a polyvalent Cre-lox tool. We rigorously tested the ability of MeuCre40 to solve tri-lox problems, by using it for the in vivo removal of neo(R)- and hprt-expression cassettes from three different tri-lox mutants.
- Published
- 2003
- Full Text
- View/download PDF
33. Intralysosomal cystine accumulation in mice lacking cystinosin, the protein defective in cystinosis.
- Author
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Cherqui S, Sevin C, Hamard G, Kalatzis V, Sich M, Pequignot MO, Gogat K, Abitbol M, Broyer M, Gubler MC, and Antignac C
- Subjects
- Alleles, Amino Acid Transport Systems, Neutral, Animals, Bone and Bones diagnostic imaging, Bone and Bones pathology, Cystinosis diagnostic imaging, Cystinosis metabolism, Cytosine metabolism, Dogs, Electroretinography, Membrane Transport Proteins, Mice, Mice, Inbred C57BL, Mice, Knockout, Models, Genetic, Promoter Regions, Genetic, Radiography, Recombinant Proteins metabolism, Retina abnormalities, Retina pathology, Reverse Transcriptase Polymerase Chain Reaction, Time Factors, Tissue Distribution, Cystine metabolism, Cystinosis genetics, Glycoproteins, Lysosomes metabolism, Membrane Proteins genetics, Membrane Proteins physiology
- Abstract
Cystinosis is an autosomal recessive disorder characterized by an accumulation of intralysosomal cystine. The causative gene, CTNS, encodes cystinosin, a seven-transmembrane-domain protein, which we recently showed to be a lysosomal cystine transporter. The most severe and frequent form of cystinosis, the infantile form, appears around 6 to 12 months, with a proximal tubulopathy (de Toni-Debré-Fanconi syndrome) and ocular damage. End-stage renal failure is reached by 10 years of age. Accumulation of cystine in all tissues eventually leads to multisystemic disease. Treatment with cysteamine, which reduces the concentration of intracellular cystine, delays disease progression but has undesirable side effects. We report the first Ctns knockout mouse model generated using a promoter trap approach. We replaced the last four Ctns exons by an internal ribosome entry site-betagal-neo cassette and showed that the truncated protein was mislocalized and nonfunctional. Ctns(-/-) mice accumulated cystine in all organs tested, and cystine crystals, pathognomonic of cystinosis, were observed. Ctns(-/-) mice developed ocular changes similar to those observed in affected individuals, bone defects and behavioral anomalies. Interestingly, Ctns(-/-) mice did not develop signs of a proximal tubulopathy, or renal failure. A preliminary therapeutic trial using an oral administration of cysteamine was carried out and demonstrated the efficiency of this treatment for cystine clearance in Ctns(-/-) mice. This animal model will prove an invaluable and unique tool for testing emerging therapeutics for cystinosis.
- Published
- 2002
- Full Text
- View/download PDF
34. Experimental IGF-I receptor deficiency generates a sexually dimorphic pattern of organ-specific growth deficits in mice, affecting fat tissue in particular.
- Author
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Holzenberger M, Hamard G, Zaoui R, Leneuve P, Ducos B, Beccavin C, Périn L, and Le Bouc Y
- Subjects
- Animals, Female, Growth Disorders genetics, Growth Disorders metabolism, Male, Mice, Mice, Transgenic, Organ Specificity, Receptor, IGF Type 1 genetics, Sex Characteristics, Sex Factors, Growth Disorders etiology, Receptor, IGF Type 1 deficiency
- Abstract
Reduced IGF type I receptor levels diminish postnatal growth rate and adult body weight in mice. Here, we studied the impact of experimental IGF receptor deficiency on tissue-specific growth by Cre-lox-mediated dosage of a floxed IGF-IR gene. We generated mice with a wide spectrum of receptor deficiency (5-82%), and separated them into two groups with either strong (> or =50%) IGF-IR deficiency (XS mice) or moderate deficiency (<50%, M mice). The growth of XS mice was significantly retarded from 3 wk after birth onward, with respect to M littermates. This effect was twice as strong in males as in females. Growth deficits persisted throughout adult life, and at 10-12 months, most organs and tissues showed specific weight defects. Skin, bone and connective tissue, muscle, spleen, heart, lung, and brain were the most severely affected organs in the XS males. With the exception of muscle and spleen, the same tissues were also significantly reduced in size in females, although to a lesser extent. The most severe growth defect, however, concerned adipose tissue. Fat pad size in XS males was only 29% (females, 44%) of M mice. The estimated number of adipocytes in XS male fat pads was only 21% that of M males (XS female, 27%). Lipid content per cell was significantly higher in XS adipocytes, whereas plasma glucose and insulin levels were low in XS males. Thus, IGF type I receptor deficiency produced mice with disproportionate postnatal organ growth, and these effects depended strongly on sex. A marked reduction in IGF-IR levels resulted in a major defect in adipose tissue.
- Published
- 2001
- Full Text
- View/download PDF
35. A targeted partial invalidation of the insulin-like growth factor I receptor gene in mice causes a postnatal growth deficit.
- Author
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Holzenberger M, Leneuve P, Hamard G, Ducos B, Perin L, Binoux M, and Le Bouc Y
- Subjects
- Animals, Animals, Newborn growth & development, Anti-Bacterial Agents, Base Sequence genetics, DNA Transposable Elements, Directed Molecular Evolution, Drug Resistance, Microbial genetics, Female, Humans, Introns genetics, Male, Mice, Molecular Sequence Data, Neomycin, Phenotype, Somatomedins metabolism, Animals, Newborn genetics, Gene Targeting, Growth Disorders genetics, RNA Splicing genetics, Receptor, IGF Type 1 genetics
- Abstract
The insulin-like growth factor (IGF) system is a major regulator of somatic growth in vertebrates. Both ligands (IGF-I and IGF-II) signal via the same IGF receptor (IGF-IR). Classical IGF-IR invalidation is lethal at birth, so that conditional models are needed to study the postnatal role of this receptor. To establish a genetically inducible invalidation of IGF-IR, we targeted the IGF-IR gene using a construct that introduced a neomycin resistance cassette into intron 2, leaving the rest of the gene intact. This neomycin resistance cassette interfered with the processing of the primary transcript, resulting in there being 12% fewer IGF-binding sites at the cell surface in heterozygous mice and 41% fewer in homozygous mice. Hetero- and homozygous offspring grew more slowly than their wild-type littermates. This difference was noticeable from 4 weeks after birth and was significant from 5 weeks after birth in males. In females, the effect on postnatal growth of insertion of the neo cassette was not significant. In males, IGF-I levels increased moderately (+26%) but significantly, indicating effective feedback regulation of the IGF system. IGF-binding protein-4 (IGFBP-4) levels, estimated by Western ligand blotting, were low in homozygotes (-38%), whereas IGFBP-1, -2, and -3 levels were unaffected. In females, IGF-I and IGFBP-1, -2, -3, and -4 levels did not differ significantly among heterozygous, homozygous, and wild-type animals. We investigated the molecular mechanism involved and characterized two RNA-splicing events that could account for the decrease in IGF-IR. The phenotype of these mice developed exclusively postnatally, and body proportions were maintained. IGF-IRneo mice constitute a new model for human postnatal growth deficiency.
- Published
- 2000
- Full Text
- View/download PDF
36. Ubiquitous postnatal LoxP recombination using a doxycycline auto-inducible Cre transgene (DAI-Cre).
- Author
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Holzenberger M, Zaoui R, Leneuve P, Hamard G, and Le Bouc Y
- Subjects
- Doxycycline pharmacology, Gene Expression Regulation, Enzymologic drug effects, Integrases genetics, Recombination, Genetic, Transgenes, Viral Proteins
- Published
- 2000
37. Effects of repetitive strains on vertebral end plates in young rats.
- Author
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Revel M, Andre-Deshays C, Roudier R, Roudier B, Hamard G, and Amor B
- Subjects
- Animals, Growth Plate ultrastructure, Intervertebral Disc pathology, Rats, Rats, Inbred Strains, Stress, Mechanical, Cumulative Trauma Disorders pathology, Growth Plate pathology, Spinal Injuries pathology
- Abstract
A histologic study of vertebral end plates was performed on young rats' tails subjected to intensive passive motion. Seven six-week-old rats had two-hour motion cycles daily, for two months. Histologic changes of proximal caudal vertebrae in the experimental group were compared with those of a control group of seven rats of the same litter which were allowed to grow freely without mechanical stress. The main findings were three striking protrusions of disk tissue into end plates, not observed in the control animals. End plate axial bulges lacking spindle-shaped cells and an increase in chondrocytes in the adjacent articular cartilage were observed. The growth-plate thickness was uneven and was absent in some areas, with disorganization of chondrocyte columns. All of these findings were present to a lesser degree in the control group and appeared to be similar to histologic changes observed in Scheuermann's disease.
- Published
- 1992
38. Effect of dystrophin gene deletions on mRNA levels and processing in Duchenne and Becker muscular dystrophies.
- Author
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Chelly J, Gilgenkrantz H, Lambert M, Hamard G, Chafey P, Récan D, Katz P, de la Chapelle A, Koenig M, and Ginjaar IB
- Subjects
- Base Sequence, Exons, Humans, Molecular Sequence Data, Muscular Dystrophies classification, Oligonucleotide Probes, RNA Splicing, Reading Frames, Restriction Mapping, Chromosome Deletion, Dystrophin genetics, Gene Amplification, Genes, Muscular Dystrophies genetics, RNA, Messenger genetics, Transcription, Genetic
- Abstract
Muscle dystrophin mRNAs from Duchenne (DMD) and Becker (BMD) patients with internal deletion of the DMD gene were quantitated and sequenced. In all cases (eight DMD and three BMD), truncated mature transcripts were found, and their amount was correlated to the clinical phenotype and to the reading frame. We focused on four cases that were apparently not in agreement with the reading frame rule. In two DMD cases, slightly reduced amounts of in-frame truncated mRNA are present but no dystrophin is detected, suggesting impaired translation and/or instability of the protein. In two BMD patients with out-of-frame deletions, the presence of minor in-frame alternatively spliced mRNA species is congruent with the observed truncated dystrophin and the mild phenotype.
- Published
- 1990
- Full Text
- View/download PDF
39. Analysis of deletions in DNA from patients with Becker and Duchenne muscular dystrophy.
- Author
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Kunkel LM, Hejtmancik JF, Caskey CT, Speer A, Monaco AP, Middlesworth W, Colletti CA, Bertelson C, Müller U, Bresnan M, Shapiro F, Tantravahi U, Speer J, Latt SA, Bartlett R, Pericak-Vance MA, Roses AD, Thompson MW, Ray PN, Worton RG, Fischbeck KH, Gallano P, Coulon M, Duros C, Boue J, Junien C, Chelly J, Hamard G, Jeanpierre M, Lambert M, Kaplan JC, Emery A, Dorkins H, McGlade S, Davies KE, Boehm C, Arveiler B, Lemaire C, Morgan GJ, Denton MJ, Amos J, Bobrow M, Benham F, Boswinkel E, Cole C, Dubowitz V, Hart K, Hodgson S, Johnson L, Walker A, Roncuzzi L, Ferlini A, Nobile C, Romeo G, Wilcox DE, Affara NA, Ferguson-Smith MA, Lindolf M, Kaariainen H, de la Chapelle A, Ionasescu V, Searby C, Ionasescu R, Bakker E, van Ommen GJ, Pearson PL, Greenberg CR, Hamerton JL, Wrogemann K, Doherty RA, Polakowska R, Hyser C, Quirk S, Thomas N, Harper JF, Darras BT, and Francke U
- Subjects
- Chromosome Mapping, DNA Restriction Enzymes metabolism, Deoxyribonuclease EcoRI, Electrophoresis, Polyacrylamide Gel, Genes, Humans, Male, Pedigree, Chromosome Deletion, DNA analysis, Deoxyribonucleases, Type II Site-Specific, Muscular Dystrophies genetics
- Abstract
Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder for which the biochemical defect is as yet unknown. Recently, two cloned segments of human X-chromosome DNA have been described which detect structural alterations within or near the genetic locus responsible for the disorder. Both of these cloned segments were described as tightly linked to the locus and were capable of detecting deletions in the DNA of boys affected with DMD. In an attempt to determine more precisely the occurrence of these deletions within a large population of DMD patients and the accuracy of one of the segments, DXS164 (pERT87), in determining the inheritance of the DMD X chromosome, the subclones 1, 8 and 15 were made available to many investigators throughout the world. Here we describe the combined results of more than 20 research laboratories with respect to the occurrence of deletions at the DXS164 locus in DNA samples isolated from patients with DMD and Becker muscular dystrophy (BMD). The results indicate that the DXS164 locus apparently recombines with DMD 5% of the time, but is probably located between independent sites of mutation which yield DMD. The breakpoints of some deletions are delineated within the DXS164 locus, and it is evident that the deletions at the DMD locus are frequent and extremely large.
- Published
- 1986
- Full Text
- View/download PDF
40. Recovery of functional DNA inserts by electroendosmotic elution during gel electrophoresis.
- Author
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Tan HV, Kitzis A, Berthollet T, Hamard G, Beldjord C, and Benarous R
- Subjects
- Buffers, DNA Restriction Enzymes, Osmosis, DNA isolation & purification, Electrophoresis instrumentation, Electrophoresis, Agar Gel instrumentation
- Abstract
In contrast to all previous preparative electrophoresis apparatus which used a pump, electroendosmotic elution uses bound electrical charges at the end of the separating gel to generate a buffer flow. The electroendosmotic flow increased with increasing currents and decreasing buffer concentrations: its exact characteristics for the built apparatus were determined. The electroendosmotic device was able to separate two DNA fragments differing in size by only 5% with a recovery over 95%. As demonstrated in practical examples of recovery and uses of DNA inserts, up to 10 micrograms of DNA per band can be loaded at a time. The recovered DNA can be used directly for nick-translation, ligation... without further treatment. The performances of the method are expected to improve still further if the charge density and pores of the electroendosmotic medium can be "made-to-order" to provide a better flow profile of the eluting buffer.
- Published
- 1988
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41. [Prenatal diagnosis of hemophilia A by analysis of DNA].
- Author
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Delpech M, Maisonneuve P, Baudis M, Deburgrave N, Hamard G, Lambert M, Bardin JM, Sultan Y, and Kaplan JC
- Subjects
- DNA Restriction Enzymes, Female, Hemophilia A genetics, Humans, Mutation, Polymorphism, Genetic, Pregnancy, DNA analysis, Hemophilia A diagnosis, Prenatal Diagnosis methods
- Abstract
Early ante-natal diagnosis of haemophilia A and the detection of female carriers is now possible in some cases by analysis of DNA. The diagnosis may be established with a 100 p. 100 reliability in subjects with large deletion by direct analysis, and in 40 p. 100 of haemophiliac families by linkage studies with the intra-genic polymorphism revealed by the restriction enzyme BcII. Intensive research indicates that this percentage will increase in the near future. In the meantime, indicative studies are possible in other families. They consist in studying extra-genic restriction polymorphism. Over 90 p. 100 of families with haemophilia A may benefit from these studies using the probes currently available. Recombination, although not yet described, remains possible, and therefore ante-natal diagnoses made by the extra-genic probe should be controlled by foetal blood sampling at the 20th week of pregnancy.
- Published
- 1986
42. [Should we still explore placental sulfatase deficiencies? Reflections apropos of a case report].
- Author
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Giovangrandi Y, Magnin G, Sauvanet E, Soutoul JH, Cedard L, Bedin M, Moraine C, Nottin P, and Hamard G
- Subjects
- Adult, Estrogens blood, Female, Humans, Ichthyosis genetics, Infant, Newborn, Male, Pedigree, Placental Insufficiency enzymology, Pregnancy, Ichthyosis diagnosis, Placenta Diseases diagnosis, Placental Insufficiency diagnosis, Sulfatases deficiency
- Abstract
The finding of low or very low levels of oestrogen during pregnancy should suggest the diagnosis of placental sulfatase deficiency, which is confirmed by performing dynamic biochemical tests. These biochemical tests also enable sulfatase deficiency to be differentiated from other conditions which may be accompanied by an abnormal decrease in oestrogens. The authors report one case and stress the harmlessness of sulfatase deficiency and the associated ichthyosis in boys and they question the value of the dehydroepiandrosterone sulfate test which only confirms an abnormality which is now well known and benign.
- Published
- 1984
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