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1.
Cell Mol Life Sci ; 65(11): 1780-90, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18480964

RESUMO

Fas-associated factor 1 (Faf1) has been described as a Fas-binding pro-apoptotic protein and as a component of the death-inducing signaling complex (DISC) in Fas-mediated apoptosis. Faf1 is able to potentiate Fas-induced apoptosis in several cell lines, although its specific functions are still not clear. Here we show that Faf1 is highly expressed in several areas of the developing telencephalon. Its expression pattern appears to be dynamic at different embryonic stages and to be progressively confined within limited territories. To decipher the specific role of Faf1 in developing brain, we used cDNA over-expression and mRNA down-regulation experiments to modulate Faf1 expression in telencephalic neural precursor cells, and we showed that in neural cell death Faf1 acts as a Fas-independent apoptotic enhancer. Moreover, we found that Faf1 protein level is down-regulated during apoptosis in a caspase- and Apaf1-dependent manner.


Assuntos
Fator Apoptótico 1 Ativador de Proteases/metabolismo , Encéfalo/embriologia , Proteínas de Transporte/metabolismo , Caspase 3/metabolismo , Neurônios/metabolismo , Células-Tronco/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Fator Apoptótico 1 Ativador de Proteases/genética , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Proteínas de Transporte/genética , Caspase 3/genética , Células Cultivadas , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Neurônios/citologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células-Tronco/citologia
2.
Ontogenez ; 38(1): 21-32, 2007.
Artigo em Russo | MEDLINE | ID: mdl-17352291

RESUMO

Among dozens of known genes, active in the developing cortex, none possessed expression strictly limited by one functional area of the cortex. We found that in 6A-99 transgenic mice, Lac-Z-reported gene was expressed selectively in the somatosensory cortex. In the primary somatosensory cortex, expression was localized in the barrel field, including the zone of representation of vibrissae, fore and hind limbs, jaws, and head. In addition, LacZ-expressing cells were found in the secondary somatosensory cortex, as well as in the nuclei of hypothalamus and dorsal and caudal raphe nuclei. In the cortex, expression of transgene 6A-99 began on day 3 of postnatal development (P3) and embraced only the area of primary somatosensory cortex: zones of representation of the snout, vibrissae, and lower jaw. On P5, pronounced expression 6A-99 was detected in the secondary somatosensory cortex and zone of representation of fore paws. Expression in the zone of representation of hind paws was observed on P7. Expression of 6A-99 in the somatosensory cortex disappeared by P50, the age of final functional maturation of the cerebral cortex. Our results suggest that the regulation of transcription in the developing mouse somatosensory cortex differs from those in other cortical areas. Transgene 6A-99 may serve as a specific molecular marker of the developing somatosensory cortex in mice and can be used for studying the mechanisms underlying genetic and epigenetic control of the neocortex functional regionalization.


Assuntos
Expressão Gênica , Córtex Somatossensorial/crescimento & desenvolvimento , Transgenes/genética , Animais , Genes Reporter/genética , Marcadores Genéticos , Óperon Lac/genética , Camundongos , Camundongos Transgênicos , beta-Galactosidase/análise , beta-Galactosidase/genética
3.
Dev Biol ; 239(2): 257-69, 2001 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-11784033

RESUMO

Cdx1 encodes a mammalian homeobox gene involved in vertebral patterning. Retinoic acid (RA) is likewise implicated in vertebral patterning. We have previously shown that Cdx1 is a direct retinoid target gene, suggesting that Cdx1 may convey some of the effects of retinoid signaling. However, RA appears to be essential for only early stages of Cdx1 expression, and therefore other factors must be involved in maintaining later stages of expression. Based on function and pattern of expression, Wnt family members, in particular Wnt3a, are candidates for regulation of expression of Cdx1. Consistent with this, we confirm prior results which demonstrated that Cdx1 can be directly regulated by Wnt signaling, and identify functional LEF/TCF response motifs essential for this response. We also find that Cdx1 expression is markedly attenuated in a stage- and tissue-specific fashion in the Wnt3a hypomorph vestigial tail, and present data demonstrating that Wnt3a and RA synergize strongly to activate Cdx1. Finally, we show that Cdx1 positively regulates its own expression. These data prompt a model whereby retinoid and Wnt signaling function directly and synergistically to initiate Cdx1 expression in the caudal embryo. Expression is then maintained, at least in part, by an autoregulatory mechanism at later stages.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Proteínas de Xenopus , Proteínas de Peixe-Zebra , Animais , Sequência de Bases , Fator de Transcrição CDX2 , Genes Reporter , Hibridização In Situ , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Técnicas de Cultura de Órgãos , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores do Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Tempo , Transfecção , Tretinoína/metabolismo , Proteínas Wnt , Proteína Wnt3 , Proteína Wnt3A , Xenopus
4.
Dev Biol ; 240(1): 46-60, 2001 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-11784046

RESUMO

Exogenous retinoic acid (RA) can evoke vertebral homeosis when administered during late gastrulation. These vertebral transformations correlate with alterations of the rostral limit of Hox gene expression in the prevertebrae, suggesting that retinoid signaling regulates the combinatorial expression of Hox genes dictating vertebral identity. Conversely, loss of certain RA receptors (RARs) results in anterior homeotic transformations principally affecting the cervical region. Despite these observations, the relationship between retinoid signaling, somitic Hox expression, and vertebral patterning is poorly understood. The members of the murine Cdx family (Cdx1, Cdx2, and Cdx4) are the homologues of Drosophila caudal and encode homeobox-containing transcription factors. Cdx1 homozygous null mutants exhibit anterior homeotic transformations, some of which are reminiscent of those in RARgamma null offspring. In Cdx1 mutants, these transformations occur concomitant with posteriorized prevertebral expression of certain Hox genes. Cdx1 has recently been demonstrated to be a direct RA target, suggesting an indirect means by which retinoid signaling may impact vertebral patterning. To further investigate this relationship, a complete allelic series of Cdx1-RARgamma mutants was generated and the skeletal phenotype assessed either following normal gestation or after administration of RA. Synergistic interactions between these null alleles were observed in compound mutants, and the full effects of exogenous RA on vertebral morphogenesis required Cdx1. These findings are consistent with a role for RA upstream of Cdx1 as regards axial patterning. However, exogenous RA attenuated several defects inherent to Cdx1 null mutants. This finding, together with the increased phenotypic severity of RARgamma-Cdx1 double null mutants relative to single nulls, suggests that these pathways also function in parallel, likely by converging on common targets.


Assuntos
Padronização Corporal/fisiologia , Proteínas de Homeodomínio/fisiologia , Receptores do Ácido Retinoico/fisiologia , Vertebrados/embriologia , Animais , Sequência de Bases , Primers do DNA , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Homeobox , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Knockout , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptor gama de Ácido Retinoico
5.
FEBS Lett ; 480(1): 63-71, 2000 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-10967330

RESUMO

The gene trap methodology is a powerful tool to characterize novel genes and analyze their importance in biological phenomena. It is based on the use of mouse embryonic stem cells and reporter vectors designed to randomly integrate into the genome, tagging an insertion site and generating a mutation. Theoretically, all the 100,000 genes present in the mouse genome could be tagged and functionally inactivated at the same time. Here we describe the basic concepts and perspectives of this methodology and show some results obtained by the gene trap approach used to study molecular cascades in basic cell biology and in developmental processes.


Assuntos
Biblioteca Gênica , Genes/fisiologia , Camundongos Transgênicos/genética , Mutagênese Insercional/métodos , Animais , Fator Apoptótico 1 Ativador de Proteases , Regulação da Expressão Gênica no Desenvolvimento , Genes/genética , Genes Reporter/genética , Vetores Genéticos/genética , Camundongos , Camundongos Transgênicos/embriologia , Camundongos Transgênicos/metabolismo , Fatores de Crescimento Neural/genética , Netrina-1 , Fenótipo , Proteínas/genética , Proteínas Supressoras de Tumor , Xenopus laevis/genética
6.
Development ; 127(17): 3805-13, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10934025

RESUMO

During mammalian development, the Cdx1 homeobox gene exhibits an early period of expression when the embryonic body axis is established, and a later period where expression is restricted to the embryonic intestinal endoderm. Cdx1 expression is maintained throughout adulthood in the proliferative cell compartment of the continuously renewed intestinal epithelium, the crypts. In this study, we provide evidence in vitro and in vivo that Cdx1 is a direct transcriptional target of the Wnt/(beta)-catenin signaling pathway. Upon Wnt stimulation, expression of Cdx1 can be induced in mouse embryonic stem (ES) cells as well as in undifferentiated rat embryonic endoderm. Tcf4-deficient mouse embryos show abrogation of Cdx1 protein in the small intestinal epithelium, making Tcf4 the likely candidate to transduce Wnt signal in this part of gut. The promoter region of the Cdx1 gene contains several Tcf-binding motifs, and these bind Tcf/Lef1/(beta)-catenin complexes and mediate (beta)-catenin-dependent transactivation. The transcriptional regulation of the homeobox gene Cdx1 in the intestinal epithelium by Wnt/(beta)-catenin signaling underlines the importance of this signaling pathway in mammalian endoderm development.


Assuntos
Proteínas Aviárias , Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Transativadores , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra , Células 3T3 , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endoderma , Genes Homeobox , Humanos , Mucosa Intestinal/metabolismo , Intestinos/embriologia , Fator 1 de Ligação ao Facilitador Linfoide , Camundongos , Ratos , Fatores de Transcrição TCF , Proteína 2 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/genética , Proteínas Wnt , beta Catenina
7.
Development ; 127(1): 13-22, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10654596

RESUMO

The morphogenetic development of the mammalian inner ear is a complex multistep process, the molecular and cellular details of which are only beginning to be unraveled. We show here that mouse netrin 1, known to be involved in axon guidance and cell migration in the central nervous system, also plays a critical morphogenetic role during semicircular canal formation. netrin 1 is expressed at high levels in the otic epithelium, in cells that will come together to form a fusion plate, a prerequisite for the formation of semicircular canals. In netrin 1 mutant mice, fusion plate formation is severely affected resulting in a reduced anterior semicircular canal and the complete lack of the posterior and lateral canals. Our results suggest that netrin 1 facilitates semicircular canal formation through two different mechanisms: (1) it participates in the detachment of the fusion plate epithelia from the basement membrane, and (2) it stimulates proliferation of the periotic mesenchymal cells which then push the epithelial cell walls together to form the fusion plate.


Assuntos
Fatores de Crescimento Neural/fisiologia , Canais Semicirculares/embriologia , Alelos , Animais , Divisão Celular , Orelha/embriologia , Epitélio/metabolismo , Feminino , Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Masculino , Mesoderma , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Netrina-1 , Proteínas Supressoras de Tumor
8.
Cell ; 94(6): 727-37, 1998 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-9753320

RESUMO

The cytosolic protein APAF1, human homolog of C. elegans CED-4, participates in the CASPASE 9 (CASP9)-dependent activation of CASP3 in the general apoptotic pathway. We have generated by gene trap a null allele of the murine Apaf1. Homozygous mutants die at embryonic day 16.5. Their phenotype includes severe craniofacial malformations, brain overgrowth, persistence of the interdigital webs, and dramatic alterations of the lens and retina. Homozygous embryonic fibroblasts exhibit reduced response to various apoptotic stimuli. In situ immunodetection shows that the absence of Apaf1 protein prevents the activation of Casp3 in vivo. In agreement with the reported function of CED-4 in C. elegans, this phenotype can be correlated with a defect of apoptosis. Our findings suggest that Apaf1 is essential for Casp3 activation in embryonic brain and is a key regulator of developmental programmed cell death in mammals.


Assuntos
Apoptose/genética , Caspases , Regulação da Expressão Gênica no Desenvolvimento , Proteínas/genética , Alelos , Animais , Fator Apoptótico 1 Ativador de Proteases , Encéfalo/enzimologia , Encéfalo/patologia , Caspase 3 , Quimera , Clonagem Molecular , Anormalidades Craniofaciais/genética , Cisteína Endopeptidases/metabolismo , Embrião de Mamíferos/anormalidades , Ativação Enzimática/fisiologia , Anormalidades do Olho , Fibroblastos/enzimologia , Humanos , Hiperplasia , Mamíferos , Camundongos , Dados de Sequência Molecular , Neurônios/química , Neurônios/enzimologia , Síndrome de Noonan/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Anormalidades da Pele
9.
Dev Dyn ; 212(2): 326-33, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9626507

RESUMO

Special vectors have been constructed that allow the trapping of genes in mouse embryonic stem (ES) cells. These vectors generally contain the neomycin phosphotransferase (neo) gene for selection and the beta-galactosidase (beta-gal) gene as a marker. Promoterless vectors can be used to identify genes that are active in undifferentiated ES cells. To also have access to genes that are inactive in totipotent ES cells, we constructed a polyadenylation (poly A) trap vector in which the expression of a poly A less neo gene is driven by a constitutive promoter, whereas the expression of beta-gal depends on the trapped sequences. We demonstrate here that this vector integrates with a high frequency into transcription units and that it traps genes with very different expression patterns in vitro and in vivo. The vector integrates efficiently into transcription units that are inactive in undifferentiated ES cells and which can be activated through in vitro differentiation. Furthermore, in vivo expression patterns demonstrate that this vector integrates into genes that exhibit a highly specific temporal and spatial expression pattern during embryogenesis.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Técnicas Genéticas , RNA Mensageiro/genética , Animais , Diferenciação Celular , Feminino , Vetores Genéticos , Canamicina Quinase/genética , Camundongos , Camundongos Transgênicos , Gravidez , Células-Tronco/citologia , beta-Galactosidase/genética
10.
Cell ; 83(4): 641-53, 1995 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-7585967

RESUMO

Cdx1 is expressed along the embryonic axis from day 7.5 postcoitum until day 12, by which time the anterior limit of expression has regressed from the hindbrain level to the forelimb bud region. To assign a functional role for Cdx1 in murine embryonic development, we have inactivated the gene via homologous recombination. Viable fertile homozygous mutant mice were obtained that show anterior homeotic transformations of vertebrae. These abnormalities were concomitant with posterior shifts of Hox gene expression domains in the somitic mesoderm. The presence of putative Cdx1-binding sites in Hox gene control regions as well as in vitro transactivation of Hoxa-7 indicates a direct regulation.


Assuntos
Proteínas Aviárias , Osso e Ossos/embriologia , Proteínas de Ligação a DNA/genética , Genes Homeobox/fisiologia , Proteínas de Homeodomínio , Animais , Animais Recém-Nascidos , Sequência de Bases , Vértebras Cervicais/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Vértebras Lombares/embriologia , Mesoderma/fisiologia , Camundongos , Dados de Sequência Molecular , Mutagênese/fisiologia , Fenótipo , Regiões Promotoras Genéticas/genética , Crânio/embriologia , Cauda/embriologia , Vértebras Torácicas/embriologia
11.
Development ; 117(1): 191-203, 1993 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7900985

RESUMO

We describe the expression pattern of the mouse Cdx-1 gene during early development, examined by both RNA and protein analyses. Cdx-1 expression began with the onset of the head process formation (day 7.5) in ectodermal and mesodermal cells of the primitive streak. Expression extended initially to the middle of the prospective hindbrain and subsequently regressed caudad to the spinal cord level by day 9.5. The mesoderm-specific expression was detected in the first somites and could be followed during their differentiation to the myotome of the dorsal somitic edge by day 12. The developing limb buds and the mesonephros exhibited expression up to day 12. No signal could be detected in notochordal cells and cells of the definitive endoderm. Thus, Cdx-1 is expressed during gastrulation when anterior-posterior positional values are established along the embryonic axes. Furthermore, the expression correlates with the formation of segmented tissue in the posterior hindbrain, the spinal cord and structures like the mesonephros.


Assuntos
Proteínas Aviárias , Sistema Nervoso Central/embriologia , Proteínas de Ligação a DNA/genética , Gástrula/fisiologia , Genes Homeobox/genética , Proteínas de Homeodomínio , Animais , Western Blotting , Ectoderma/fisiologia , Extremidades/embriologia , Expressão Gênica/genética , Imuno-Histoquímica , Hibridização In Situ , Mesoderma/fisiologia , Mesonefro/fisiologia , Camundongos
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