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1.
Nat Commun ; 5: 4563, 2014 Aug 01.
Article in English | MEDLINE | ID: mdl-25081057

ABSTRACT

Tuberous sclerosis complex (TSC), caused by dominant mutations in either TSC1 or TSC2 tumour suppressor genes is characterized by the presence of brain malformations, the cortical tubers that are thought to contribute to the generation of pharmacoresistant epilepsy. Here we report that tuberless heterozygote Tsc1(+/-) mice show functional upregulation of cortical GluN2C-containing N-methyl-D-aspartate receptors (NMDARs) in an mTOR-dependent manner and exhibit recurrent, unprovoked seizures during early postnatal life (

Subject(s)
Anticonvulsants/pharmacology , Epilepsy/drug therapy , Pyrazoles/pharmacology , Quinolones/pharmacology , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , TOR Serine-Threonine Kinases/genetics , Tuberous Sclerosis/drug therapy , Tumor Suppressor Proteins/genetics , Action Potentials/drug effects , Animals , Disease Models, Animal , Electroencephalography , Epilepsy/genetics , Epilepsy/metabolism , Epilepsy/pathology , Gene Expression Regulation , Heterozygote , Humans , Male , Mice , Mice, Transgenic , Microtomy , Neocortex/drug effects , Neocortex/metabolism , Neocortex/pathology , Patch-Clamp Techniques , Receptors, N-Methyl-D-Aspartate/genetics , Receptors, N-Methyl-D-Aspartate/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Tissue Culture Techniques , Tuberous Sclerosis/genetics , Tuberous Sclerosis/metabolism , Tuberous Sclerosis/pathology , Tuberous Sclerosis Complex 1 Protein , Tumor Suppressor Proteins/deficiency
2.
J Immunol ; 165(3): 1364-73, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10903739

ABSTRACT

T cell differentiation in the mouse thymus is an intricate, highly coordinated process that requires the assembly of TCR complexes from individual components, including those produced by the precisely timed V(D)J recombination of TCR genes. Mice carrying a homozygous deletion of the TCR beta transcriptional enhancer (E beta) demonstrate an inhibition of V(D)J recombination at the targeted TCR beta locus and a block in alpha beta T cell differentiation. In this study, we have characterized the T cell developmental defects resulting from the E beta-/- mutation, in light of previously reported results of the analyses of TCR beta-deficient (TCR beta-/-) mice. Similar to the latter mice, production of TCR beta-chains is abolished in the E beta-/- animals, and under these conditions differentiation into cell-surface TCR-, CD4+CD8+ double positive (DP) thymocytes depends essentially on the cell-autonomous expression of TCR delta-chains and, most likely, TCR gamma-chains. However, contrary to previous reports using TCR beta-/- mice, a minor population of TCR gamma delta+ DP thymocytes was found within the E beta-/- thymi, which differ in terms of T cell-specific gene expression and V(D)J recombinase activity, from the majority of TCR-, alpha beta lineage-committed DP thymocytes. We discuss these data with respect to the functional role of E beta in driving alpha beta T cell differentiation and the mechanism of alpha beta T lineage commitment.


Subject(s)
Enhancer Elements, Genetic/immunology , Gene Deletion , Genes, T-Cell Receptor beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/metabolism , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Lineage/genetics , Cell Lineage/immunology , Enhancer Elements, Genetic/genetics , Flow Cytometry , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Gene Rearrangement, delta-Chain T-Cell Antigen Receptor/genetics , Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor/genetics , Genes, T-Cell Receptor delta/genetics , Genes, T-Cell Receptor gamma/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Receptors, Antigen, T-Cell, alpha-beta/biosynthesis , Receptors, Antigen, T-Cell, alpha-beta/deficiency , Receptors, Antigen, T-Cell, gamma-delta/biosynthesis , Receptors, Antigen, T-Cell, gamma-delta/genetics , T-Lymphocyte Subsets/immunology , Thymus Gland/cytology , Thymus Gland/immunology , Thymus Gland/metabolism
3.
Hum Mol Genet ; 8(13): 2497-505, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10556298

ABSTRACT

Prader-Willi syndrome (PWS) is a complex neurogenetic disorder. The phenotype is likely to be a contiguous gene syndrome involving genes which are paternally expressed only, located in the human 15q11-q13 region. Four mouse models of PWS have been reported but these do not definitively allow the delineation of the critical region and the associated genes involved in the aetiology of PWS. Moreover, targeted mutagenesis of mouse homologues of the human candidate PWS genes does not appear to result in any of the features of PWS. Therefore, the isolation of new genes in this region remains crucial for a better understanding of the molecular basis of PWS. In this manuscript, we report the characterization of MAGEL2 and its mouse homologue Magel2. These are located in the human 15q11-q13 and mouse 7C regions, in close proximity to NDN / Ndn. By northern blot analysis we did not detect any expression of MAGEL2 / Magel2 but by RT-PCR analysis, specific expression was detected in fetal and adult brain and in placenta. Both genes are intronless with tandem direct repeat sequences contained within a CpG island in the 5'-untranscribed region. The transcripts encode putative proteins that are homologous to the MAGE proteins and NDN. Moreover, MAGEL2 / Magel2 are expressed only from the paternal allele in brain, suggesting a potential role in the aetiology of PWS and its mouse model, respectively.


Subject(s)
Chromosome Mapping , Prader-Willi Syndrome/genetics , Proteins/genetics , 5' Untranslated Regions , Amino Acid Sequence , Animals , Antigens, Neoplasm , Base Sequence , Brain/metabolism , Chromosomes, Human, Pair 15 , CpG Islands , Gene Expression , Genomic Imprinting , Humans , Mice , Mice, Inbred Strains , Molecular Sequence Data , Pedigree , Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Tandem Repeat Sequences
4.
Eur J Hum Genet ; 5(5): 324-32, 1997.
Article in English | MEDLINE | ID: mdl-9412790

ABSTRACT

Prader-Willi syndrome (PWS) is a neurogenetic disorder resulting from the loss of paternal expression of gene(s) localized in the 15q11-q12 region. A new human gene encoding a putative protein with high homology to the mouse NECDIN protein has recently been characterized and mapped to chromosome 15q11-q12. It is expressed from the paternal allele only, suggesting its potential involvement in PWS. We now report the localization of the mouse Necdin gene in a region of conserved synteny to the human PWS region. We demonstrate the paternal specific expression of Necdin in the mouse central nervous system, and show that parental alleles display a differential methylation profile in the coding region. Finally, fluorescence in situ hybridization analysis reveals an asynchronous pattern of replication at the Necdin locus. These results clearly demonstrate imprinting of the mouse Necdin gene. Mouse models will be powerful tools in the study of human PWS phenotype and imprinting mechanisms.


Subject(s)
Chromosome Mapping , Gene Expression Regulation, Developmental/genetics , Genomic Imprinting/genetics , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Prader-Willi Syndrome/genetics , Alleles , Animals , Base Sequence , Brain , Brain Chemistry , Conserved Sequence/genetics , Crosses, Genetic , DNA Methylation , DNA Replication , Female , Fetus , Humans , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Muridae , RNA, Messenger/analysis
5.
Proc Natl Acad Sci U S A ; 93(15): 7877-81, 1996 Jul 23.
Article in English | MEDLINE | ID: mdl-8755570

ABSTRACT

Intrathymic T-cell development requires temporally regulated rearrangement and expression of T-cell receptor (TCR) genes. To assess the role of the TCR beta gene transcriptional enhancer (Ebeta) in this process, mouse strains in which Ebeta is deleted were generated using homologous recombination techniques. We report that mice homozygous for the Ebeta deletion, whether a selectable marker gene is present or not, show a block in alphabeta T-cell development at the CD4-CD8- double-negative cell stage, whereas the number of gammadelta+ T cells is normal, few CD4+CD8+ double-positive thymocytes and no alphabeta+ T cells are produced. DNA-PCR and RNA-PCR analyses of thymic cells from homozygous mutants showed no evidence of TCR beta gene rearrangement although germ-line Vbeta transcripts were detected at a low level, in heterozygous T cells, the targeted allele is not rearranged. Thus, deletion of Ebeta totally prevents rearrangement, but not transcription, of the targeted beta locus. These data formally establish the critical role played by Ebeta in cis-activation of the TCR beta locus for V(D)J recombination during alphabeta T-cell development.


Subject(s)
Aging/immunology , Enhancer Elements, Genetic , Gene Deletion , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocytes/immunology , Transcription, Genetic , Animals , Flow Cytometry , Genotype , Lymph Nodes/growth & development , Lymph Nodes/immunology , Mice , Mice, Knockout , Polymerase Chain Reaction , Receptors, Antigen, T-Cell, alpha-beta/biosynthesis , Spleen/growth & development , Spleen/immunology , Stem Cells , T-Lymphocyte Subsets/immunology , Thymus Gland/growth & development , Thymus Gland/immunology
6.
EMBO J ; 12(11): 4335-46, 1993 Nov.
Article in English | MEDLINE | ID: mdl-8223443

ABSTRACT

We describe transgenic mice carrying germline variable gene segments associated with either the T cell receptor (TCR) beta or alpha gene enhancers (E beta or E alpha). Transgenic constructs underwent high rates of site-specific rearrangements predominantly in T cells from independent mice. Rearrangements of the E beta-containing transgenes began at different stages of T cell differentiation in embryonic and adult thymus than did the E alpha-containing ones, with a pattern superimposable upon the patterns of TCR beta or TCR alpha gene expression, respectively. We demonstrate that sequences within the TCR beta and TCR alpha gene enhancers confer tissue- and stage-specificity upon the V(D)J recombination events affecting adjacent gene segments. The patterns of transgene expression also gave information on developmental events and lineage relationships (gamma delta versus alpha beta) during T cell development.


Subject(s)
Enhancer Elements, Genetic/genetics , Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , Receptors, Antigen, T-Cell, alpha-beta/genetics , Recombination, Genetic , Aging , Animals , B-Lymphocytes/immunology , DNA Nucleotidyltransferases/metabolism , Hematopoietic Stem Cells/immunology , Lymph Nodes/immunology , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , T-Lymphocyte Subsets/immunology , T-Lymphocytes/immunology , Thymus Gland/embryology , Thymus Gland/immunology , VDJ Recombinases
7.
Exp Cell Res ; 206(2): 212-9, 1993 Jun.
Article in English | MEDLINE | ID: mdl-7684693

ABSTRACT

C-abl and c-mos, two proto-oncogenes with unique patterns of expression in gonadal tissues, were examined by in situ hybridization with respect to their expression in the germ line. C-abl transcripts were observed to be most abundant in late round spermatids and elongating spermatids. C-mos transcripts were also expressed in round spermatids, but at much reduced levels compared to the levels of c-abl. C-abl mRNAs were also detected in mouse oocytes which have entered the growth phase. This temporal specificity of accumulation of c-abl transcripts was similar to that observed for c-mos, which also accumulates in growing and fully grown oocytes (follicle stages 4-6). Quantitative evaluation suggested that c-abl mRNA levels in oocytes are at least an order of magnitude lower than those of c-mos transcripts.


Subject(s)
Gene Expression Regulation , Genes, abl , Genes, mos , Oocytes/physiology , Ovary/physiology , Spermatocytes/physiology , Testis/physiology , Aging , Animals , Antisense Elements (Genetics) , Blotting, Northern , Female , In Situ Hybridization , Male , Mice , Oligonucleotide Probes , Oocytes/cytology , Ovary/cytology , Ovary/growth & development , Poly A/genetics , Poly A/isolation & purification , RNA/genetics , RNA/isolation & purification , RNA, Messenger/analysis , RNA, Messenger/genetics , Spermatocytes/cytology , Testis/cytology , Testis/growth & development , Transcription, Genetic
8.
Dev Biol ; 156(1): 136-45, 1993 Mar.
Article in English | MEDLINE | ID: mdl-7680626

ABSTRACT

A possible correlation between the structural organization of homeobox-containing genes and their cell-specific expression was examined in studies determining similarities and differences in the expression patterns of orthologues and paralogues of the murine Hox-1.4 gene. We first compared the expression pattern of members of the Hox-1.4 subfamily, Hox-2.6 and Hox-4.2, in the adult mouse testis. Although these three evolutionarily related genes exhibited similar anterior limits of expression in the embryonic central nervous system, their cellular specificity of expression was very different in the adult testis. Hox-1.4 was abundantly expressed only in the germ cells; Hox-2.6 was expressed at very low levels in both spermatogenic cells and somatic cells; and Hox-4.2 transcripts appeared to be restricted to the somatic cells. We next analyzed the expression of several of the Hox-1.4 orthologues in the mouse testis to determine if other members of the Hox-1 cluster are involved in the male germ cell differentiation pathway. We showed that the two adjacent genes of Hox-1.4, Hox-1.3 and Hox-1.5, are expressed in the germ line but at lower levels. Further, both Hox-1.3 and Hox-1.4 produced unique, germ line-specific transcripts as compared to other adult tissues and the Day 12.5 embryo.


Subject(s)
Genes, Homeobox , Spermatozoa/physiology , Testis/physiology , Transcription, Genetic , Animals , Biological Evolution , Blotting, Northern , DNA Probes , Embryo, Mammalian/physiology , Male , Mice , Poly A/analysis , Poly A/genetics , RNA/analysis , RNA/genetics , RNA Probes , RNA, Messenger , Restriction Mapping , Spermatozoa/cytology , Testis/cytology , Testis/growth & development
9.
Cell Growth Differ ; 2(2): 77-83, 1991 Feb.
Article in English | MEDLINE | ID: mdl-2069871

ABSTRACT

We have cloned a murine transforming growth factor (TGF) beta 3 complementary (cDNA) from normal tissue by low stringency screening of a testicular cDNA library with a TGF beta 1 probe. The coding domain of this TGF beta 3 cDNA agrees completely with the sequence reported for the TGF beta 3 cDNA isolated from the AKR-2B cell line, but the testicular clone uses a distinct and unusual polyadenylation signal resulting in an altered 3' untranslated domain. Northern blot hybridization analysis of gonadal tissues showed that both TGF beta 3 and TGF beta 1 mRNAs are detectable in the mouse testis and ovary. A detailed analysis of TGF beta 3 and TGF beta 1 gene expression in normal and germ cell-deficient male mice showed that the somatic cell compartment of the mouse testis expresses the usual-sized transcripts for both genes. However, a smaller (1.8-kilobase) TGF beta 1 mRNA is expressed selectively in male germ cells, and expression of this transcript was constitutive throughout the spermatogenic stages examined. This result demonstrates a new pattern of TGF beta 1 gene expression, consistent with cell lineage-specific transcriptional regulation during spermatogenesis.


Subject(s)
Transforming Growth Factor beta/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA/genetics , Female , Gene Expression Regulation , Kidney , Lung , Male , Mice , Mice, Inbred AKR , Molecular Sequence Data , Organ Specificity , Ovary , Spermatogenesis , Testis
12.
Reprod Nutr Dev (1980) ; 28(4B): 1113-28, 1988.
Article in English | MEDLINE | ID: mdl-3244905

ABSTRACT

In 13 and 14-day old fetal rat ovaries maintained 3 to 10 days in organ culture, purified bovine anti-Müllerian hormone (AMH) (1.5 to 3 micrograms/ml) induced a characteristic freemartin effect. Gonadal volume and germ cell number were significantly reduced, compared to control ovaries cultured in anhormonal medium, and epithelial cells with large clear cytoplasm linked by interdigitations differentiated in the gonadal blastema. These cells resembling rat fetal Sertoli cells became polarized and formed seminiferous cord-like structures delineated by a basal membrane containing laminin and fibronectin as is the case of testicular seminiferous cords at the first step of their differentiation. These data indicate that AMH is probably the testicular factor responsible for the morphological modifications of bovine freemartin gonads and suggest that this hormone could also be involved in normal morphological differentiation of the testis. In contrast, in fetal rat ovaries, AMH did not trigger the testosterone production which occurs in freemartin gonads at an early stage of the gestation.


Subject(s)
Embryonic and Fetal Development/drug effects , Freemartinism/physiopathology , Glycoproteins , Growth Inhibitors , Mullerian Ducts/physiology , Ovary/embryology , Testicular Hormones/pharmacology , Animals , Anti-Mullerian Hormone , Female , Organ Culture Techniques , Ovary/anatomy & histology , Ovary/drug effects , Rats , Rats, Inbred Strains
13.
Development ; 100(1): 43-55, 1987 May.
Article in English | MEDLINE | ID: mdl-3652967

ABSTRACT

To determine whether anti-Müllerian hormone (AMH) is responsible for the gonadal lesions observed in bovine genetic females united by placental anastomoses to male twins (freemartins), prospective ovaries of fetal rats were exposed to purified bovine AMH in vitro. In cultures initiated at 14 days p.c. and maintained 3 to 10 days, AMH consistently induced a characteristic 'freemartin effect', namely reduction of gonadal volume, germ cell depletion and differentiation, in the gonadal blastema, of epithelial cells with large clear cytoplasm linked by interdigitations, resembling rat fetal Sertoli cells. These cells tend to become polarized and form cords, delineated by a continuous basal membrane containing laminin and fibronectin. Such structures, resembling developing seminiferous cords, were not detected in control ovarian cultures. These data strongly suggest that AMH is the testicular factor responsible for triggering the morphological abnormalities of freemartin gonads.


Subject(s)
Freemartinism/etiology , Glycoproteins , Growth Inhibitors , Ovary/drug effects , Testicular Hormones/pharmacology , Animals , Anti-Mullerian Hormone , Cell Count/drug effects , Dose-Response Relationship, Drug , Female , Freemartinism/pathology , Mullerian Ducts/drug effects , Organ Culture Techniques , Ovary/embryology , Ovary/ultrastructure , Rats , Rats, Inbred Strains
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