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1.
PLoS Genet ; 16(1): e1008577, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31929527

RESUMO

Circadian systems provide a fitness advantage to organisms by allowing them to adapt to daily changes of environmental cues, such as light/dark cycles. The molecular mechanism underlying the circadian clock has been well characterized. However, how internal circadian clocks are entrained with regular daily light/dark cycles remains unclear. By collecting and analyzing indirect calorimetry (IC) data from more than 2000 wild-type mice available from the International Mouse Phenotyping Consortium (IMPC), we show that the onset time and peak phase of activity and food intake rhythms are reliable parameters for screening defects of circadian misalignment. We developed a machine learning algorithm to quantify these two parameters in our misalignment screen (SyncScreener) with existing datasets and used it to screen 750 mutant mouse lines from five IMPC phenotyping centres. Mutants of five genes (Slc7a11, Rhbdl1, Spop, Ctc1 and Oxtr) were found to be associated with altered patterns of activity or food intake. By further studying the Slc7a11tm1a/tm1a mice, we confirmed its advanced activity phase phenotype in response to a simulated jetlag and skeleton photoperiod stimuli. Disruption of Slc7a11 affected the intercellular communication in the suprachiasmatic nucleus, suggesting a defect in synchronization of clock neurons. Our study has established a systematic phenotype analysis approach that can be used to uncover the mechanism of circadian entrainment in mice.


Assuntos
Ritmo Circadiano/genética , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Aprendizado de Máquina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Receptores de Ocitocina/genética , Proteínas Repressoras/genética , Serina Endopeptidases/genética , Proteínas de Ligação a Telômeros/genética , Complexos Ubiquitina-Proteína Ligase/genética
2.
Mamm Genome ; 33(1): 192-202, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34482437

RESUMO

The Asian Mouse Mutagenesis Resource Association (AMMRA) is a non-profit organization consisting of major resource and research institutions with rodent expertise from within the Asia Pacific region. For more than a decade, aiming to support biomedical research and stimulate international collaboration, AMMRA has always been a friendly and passionate ally of Asian and Australian member institutions devoted to sharing knowledge, exchanging resources, and promoting biomedical research. AMMRA is also missioned to global connection by working closely with the consortiums such as the International Mouse Phenotyping Consortium and the International Mouse Strain Resource. This review discusses the emergence of AMMRA and outlines its many roles and responsibilities in promoting, assisting, enriching research, and ultimately enhancing global life science research quality.


Assuntos
Animais de Laboratório , Pesquisa Biomédica , Animais , Ásia , Austrália , Camundongos , Mutagênese
3.
Dev Dyn ; 240(5): 1289-302, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21465622

RESUMO

Sonic hedgehog (Shh) signaling in the limb plays a central role in coordination of limb patterning and outgrowth. Shh expression in the limb is limited to the cells of the zone of polarizing activity (ZPA), located in posterior limb bud mesoderm. Shh is not expressed by limb ectoderm or apical ectodermal ridge (AER), but recent studies suggest a role for AER-Shh signaling in limb patterning. Here, we have examined the effects of activation of Shh signaling in the AER. We find that targeted expression of Shh in the AER activates constitutive Shh signaling throughout the AER and subjacent limb mesoderm, and causes a range of limb patterning defects with progressive severity from mild polydactyly, to polysyndactyly with proximal defects, to severe oligodactyly with phocomelia and partial limb ventralization. Our studies emphasize the importance of control of the timing, level and location of Shh pathway signaling for limb anterior-posterior, proximal-distal, and dorsal-ventral patterning.


Assuntos
Extremidades/embriologia , Proteínas Hedgehog/metabolismo , Botões de Extremidades/metabolismo , Animais , Ectoderma/embriologia , Ectoderma/metabolismo , Proteínas Hedgehog/genética , Botões de Extremidades/embriologia , Camundongos , Camundongos Transgênicos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
4.
PLoS One ; 17(5): e0264934, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35522651

RESUMO

Atherosclerosis is the main cause of cardiac and peripheral vessel infarction in developed countries. Recent studies have established that gut microbiota and their metabolites play important roles in the development and progression of cardiovascular disease; however, the underlying mechanisms remain unclear. The present study aimed to investigate endothelium plaque lesion formation in ApoE-deficient rats fed a normal chow diet under germ-free (GF) and specific-pathogen-free (SPF) conditions at various time points. There was no difference in serum cholesterol and triglyceride levels between SPF-rats and GF-rats. Histological studies revealed that the GF-rats developed endothelium plaques in the aorta from 26 to 52 weeks, but this was not observed in SPF-rats. GF-rat coronary arteries had moderate-to-severe endothelium lesions during this time period, but SPF-rat coronary arteries had only mild lesion formation. Immunohistochemical staining showed higher accumulation of CD68-positive and arginase-negative foamy-like macrophages on the arterial walls of GF-rats, and expression of TNF-α and IL-6 in foam cells was only observed in GF-rats. In addition, microbial metabolites, including equol derivatives, enterolactone derivatives, indole-3-propionate, indole-3-acrylic acid, cholic acid, hippuric acid, and isoquinolone, were significantly higher in the SPF group than in the GF group. In conclusion, our results indicate that gut microbiota may attenuate atherosclerosis development.


Assuntos
Aterosclerose , Microbioma Gastrointestinal , Placa Aterosclerótica , Animais , Apolipoproteínas E/genética , Endotélio , Indóis , Ratos
5.
J Pathol ; 222(3): 238-48, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20814903

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common life-threatening inherited diseases, and the PKD1 gene is responsible for most cases of this disease. Previous efforts to establish a mouse model that recapitulates the phenotypic characteristics of ADPKD, which have used conventional or conditional knockout of the mouse orthologue Pkd1, have been unsuccessful or unreliable. In a previous study, we described the generation of a novel Pkd1 hypomorphic allele, in which Pkd1 expression was significantly reduced but not totally blocked. These Pkd1 homozygous mutant mice rapidly developed renal cystic disease, supporting the hypothesis that 'haploinsufficiency' explains development of the ADPKD phenotype. In the present study, we further investigated the Pkd1 haploinsufficiency effect by generating Pkd1 knockdown transgenic mice with co-cistronic expression of two miRNA hairpins specific to Pkd1 transcript and an Emerald GFP reporter driven by a human ubiquitin B promoter. Two transgenic lines which had ∼60-70% reduction of Pkd1 expression developed severe renal cystic disease at a rate similar to that of human ADPKD. These results further support the haploinsufficiency hypothesis, and suggest that the onset and progression of the renal cystic diseases are correlated with the level of Pkd1 expression. The two novel mutant lines of mice appear to be ideal models for the study of ADPKD.


Assuntos
Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Animais , Apoptose , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Células Epiteliais/patologia , Técnicas de Silenciamento de Genes/métodos , Túbulos Renais/patologia , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Canais de Cátion TRPP/metabolismo
6.
J Immunol ; 183(4): 2373-81, 2009 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-19605695

RESUMO

Translationally controlled tumor protein (TCTP) is expressed throughout T cell development and prominently induced following T cell activation. However, its function(s) during these processes is unclear. Here, we demonstrated that conditional deletion of TCTP before the beta selection checkpoint resulted into a partial block of thymocyte development at the double-negative (DN) 3 stage. Deletion of TCTP in the double-positive (DP) stage did not cause any significant phenotype in the thymus except a slight increase of mature CD8 single-positive (SP) thymocytes. In contrast to the very modest phenotype observed in the thymus, a significant reduction of mature T cells was observed in the peripheral lymphoid organs of these two conditional null TCTP mutant mice. Detailed analysis revealed that the latter phenotype (peripheral T cell lymphopenia) was largely due to a decreased viability of mature TCTP-deficient (TCTP(-/-)) T cells. Transgenic expression of the anti-apoptotic protein Bcl-2 rescued the partial block of early thymocyte development, but not peripheral T cell lymphopenia of T-lineage-specific TCTP(-/-) mice, suggesting that the signaling networks of TCTP in these two processes are not identical. Last, we demonstrated that TCTP(-/-) T cells manifested a significant defect in T cell Ag receptor (TCR)-mediated cell proliferation. Further analysis revealed that such defect was due to a marked delay in the initial cell-cycle entry of TCTP(-/-) T cells following TCR stimulation. Together, these results indicate that TCTP plays a very modest role in thymocyte development, but is critical for peripheral T cell maintenance and TCR-mediated cell proliferation.


Assuntos
Biomarcadores Tumorais/fisiologia , Proliferação de Células , Homeostase/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Biomarcadores Tumorais/deficiência , Biomarcadores Tumorais/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Homeostase/genética , Imunofenotipagem , Ligantes , Linfopenia/genética , Linfopenia/imunologia , Linfopenia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/citologia , Timo/citologia , Timo/imunologia , Timo/metabolismo , Proteína Tumoral 1 Controlada por Tradução
7.
Hum Mol Genet ; 17(21): 3368-79, 2008 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-18684731

RESUMO

Juvenile nephronophthisis type I is the most common genetic disorder causing end-stage renal failure in children and young adults. The defective gene responsible has been identified as NPHP1. Its gene product, nephrocystin-1, is a novel protein of uncertain function that is widely expressed in many tissues and not just confined to the kidney. To gain insight into the physiological function of nephrocystin, Nphp1-targeted mutant mice were generated by homologous recombination. Interestingly, homozygous Nphp1 mutant mice were viable without renal manifestations of nephronophthisis. They appeared normal, but males were infertile with oligoteratozoospermia. Histological analysis of the seminiferous tubules showed that spermatogenesis was blocked at the early stages of spermatid elongation, with degenerating spermatids sloughing off into the lumen. Electron microscopic analysis revealed detachment of early elongating spermatids from Sertoli cells, and a failure of sperm head and tail morphogenesis. However, a few mature spermatozoa were still deposited in the epididymis, though they were frequently dead, immotile, or malformed. These novel findings indicate that nephrocystin is critically required for the differentiation of early elongating spermatids into spermatozoa in mice. The possible roles of nephrocystin in the formation and maintenance of Sertoli-spermatid junctions are still under investigation.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Infertilidade Masculina/genética , Espermatogênese/genética , Espermatozoides/crescimento & desenvolvimento , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Peso Corporal , Proteínas do Citoesqueleto , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Nefropatias/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Tamanho do Órgão , Fenótipo , Túbulos Seminíferos/ultraestrutura , Contagem de Espermatozoides , Espermatozoides/diagnóstico por imagem , Espermatozoides/ultraestrutura , Ultrassonografia
8.
Nat Commun ; 9(1): 288, 2018 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-29348434

RESUMO

Metabolic diseases are a worldwide problem but the underlying genetic factors and their relevance to metabolic disease remain incompletely understood. Genome-wide research is needed to characterize so-far unannotated mammalian metabolic genes. Here, we generate and analyze metabolic phenotypic data of 2016 knockout mouse strains under the aegis of the International Mouse Phenotyping Consortium (IMPC) and find 974 gene knockouts with strong metabolic phenotypes. 429 of those had no previous link to metabolism and 51 genes remain functionally completely unannotated. We compared human orthologues of these uncharacterized genes in five GWAS consortia and indeed 23 candidate genes are associated with metabolic disease. We further identify common regulatory elements in promoters of candidate genes. As each regulatory element is composed of several transcription factor binding sites, our data reveal an extensive metabolic phenotype-associated network of co-regulated genes. Our systematic mouse phenotype analysis thus paves the way for full functional annotation of the genome.


Assuntos
Metabolismo Basal/genética , Glicemia/metabolismo , Peso Corporal/genética , Diabetes Mellitus Tipo 2/genética , Obesidade/genética , Consumo de Oxigênio/genética , Triglicerídeos/metabolismo , Animais , Área Sob a Curva , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Ensaios de Triagem em Larga Escala , Humanos , Doenças Metabólicas/genética , Camundongos , Camundongos Knockout , Fenótipo
9.
Endocrinology ; 148(4): 1804-12, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17218410

RESUMO

The CYP11A1 encodes cytochrome P450scc, catalyzing the first step of steroidogenesis in adrenals and gonads under the control of cAMP-mediated hormonal signals. The cAMP-induced activation of human CYP11A1 has been suggested to depend on the transcription factor cAMP-responsive element-binding protein (CREB), but the CREB action cannot explain the chronic cAMP effect on CYP11A1 activation. To further understand the mechanism of human CYP11A1 activation, we dissected the functions of the upstream cAMP responsive sequences (U-CRS) containing a core sequence, U identical to the steroidogenic factor-1 (SF-1)-binding site, and two flanking TPA-responsive element/cAMP-responsive element-like elements, C1 and C2. The EMSA assays showed that the binding activities of U with SF-1 as well as C1 or C2 with activating protein-1 (AP-1)/CREB-like proteins are induced by cAMP. The results from the site-directed mutagenesis analyses revealed that all three elements are required for the U-CRS function and any mutation of C1, C2, or U impairs the response to cAMP stimulation. In transgenic mice, the single or double mutations of C1 and C2 resulted in the reduction of reporter gene expression accompanied with poor hormonal response. The cAMP induction on the U-CRS activity was mimicked and enhanced by the overexpressed c-Jun in the presence of SF-1, but was abolished by the overexpression of an AP-1 dominant-negative mutant, FosB2. Furthermore, we have observed the interdependent transactivation between SF-1 and c-Jun on the U-CRS function. These results collectively demonstrate that SF-1 and AP-1 cooperate to activate CYP11A1 transcription in vitro and in vivo.


Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , AMP Cíclico/farmacologia , Proteínas de Homeodomínio/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Fator de Transcrição AP-1/fisiologia , Fatores de Transcrição/fisiologia , Animais , Células Cultivadas , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Receptores Citoplasmáticos e Nucleares/metabolismo , Elementos Reguladores de Transcrição , Fator Esteroidogênico 1 , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , Transfecção
10.
Mol Endocrinol ; 16(8): 1943-50, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12145347

RESUMO

Steroid deficiencies are diseases affecting salt levels, sugar levels, and sexual differentiation. To study steroid deficiency in more detail, we used a gene-targeting technique to insert a neo gene into the first exon to disrupt Cyp11a1, the first gene in steroid biosynthetic pathways. Cyp11a1 null mice do not synthesize steroids. They die shortly after birth, but can be rescued by steroid injection. Due to the lack of feedback inhibition by glucocorticoid, their circulating ACTH levels are exceedingly high; this results in ectopic Cyp21 gene expression in the testis. Male Cyp11a1 null mice are feminized with female external genitalia and underdeveloped male accessory sex organs. Their testis, epididymis, and vas deferens are present, but undersized. In addition, their adrenals and gonads accumulate excessive amounts of lipid. The lack of steroid production, abnormal gene expression, and aberrant reproductive organ development resemble various steroid deficiency syndromes, making these mice good models for studies of steroid function and regulation.


Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol/deficiência , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Esteroides/biossíntese , Glândulas Suprarrenais/anormalidades , Animais , Sistema Enzimático do Citocromo P-450/genética , Eletrólitos/metabolismo , Feminização/genética , Feminização/metabolismo , Expressão Gênica , Marcação de Genes , Genitália Masculina/anormalidades , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Knockout , Esteroide 21-Hidroxilase , Síndrome
12.
Genesis ; 45(2): 59-65, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17216615

RESUMO

The first step of steroid biosynthesis is catalyzed by cytochrome P450scc, encoded by CYP11A1. To achieve steroidogenic tissue-specific inactivation of genes in vivo by the Cre-loxP approach, we used the 4.4-kb regulatory region of the human CYP11A1 gene to drive Cre recombinase expression in the tissues that produce steroids. The resulting SCC-Cre mice express high levels of Cre in the adrenal cortex and gonads at the same sites as that for the endogenous CYP11A1 expression. In addition, Cre activity was found in the diencephalon and midbrain. In the developing brain, the Cre activity was first detected in the embryonic day 10.5. Our study is the first to show that the 4.4-kb CYP11A1 promoter is transcriptionally active in the brain in vivo.


Assuntos
Glândulas Suprarrenais/metabolismo , Encéfalo/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Integrases/genética , Ovário/metabolismo , Regiões Promotoras Genéticas , Testículo/metabolismo , Animais , Feminino , Humanos , Integrases/biossíntese , Masculino , Camundongos , Camundongos Transgênicos
13.
Dev Growth Differ ; 49(6): 515-21, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17555518

RESUMO

The homeodomain transcription factor Dlx5 has been implicated in the regulation of chondrocyte and osteoblast differentiation during endochondral ossification in the developing limb. In a gain-of-function approach to directly investigate the role of Dlx5 in chondrocyte maturation, we have used cartilage-specific Col2a1-Dlx5 promoter/enhancer constructs to target overexpression of Dlx5 to the differentiating cartilage models of the limbs of transgenic mice. Targeted overexpression of Dlx5 in cartilage rudiments results in the formation of shortened skeletal elements containing excessive numbers of hypertrophic chondrocytes and expanded domains of expression of Ihh and type X collagen, molecular markers of hypertrophic maturation. This suggests that hypertrophic differentiation is enhanced in response to Dlx5 misexpression. Skeletal elements overexpressing Dlx5 also exhibit a marked reduction in the zone of proliferation, indicating that overexpression of Dlx5 reduces chondrocyte proliferation concomitant with promoting hypertrophic maturation. Taken together these results indicate that Dlx5 is a positive regulator of chondrocyte maturation during endochondral ossification, and suggest that it regulates the process at least in part by promoting the conversion of immature proliferating chondrocytes into hypertrophying chondrocytes; a critical step in the maturation process.


Assuntos
Desenvolvimento Ósseo/fisiologia , Diferenciação Celular/fisiologia , Condrócitos/citologia , Extremidades/embriologia , Proteínas de Homeodomínio/fisiologia , Animais , Desenvolvimento Ósseo/genética , Diferenciação Celular/genética , Condrócitos/patologia , Colágeno Tipo II/genética , Extremidades/anatomia & histologia , Proteínas de Homeodomínio/genética , Hipertrofia , Deformidades Congênitas dos Membros/embriologia , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
14.
Am J Pathol ; 168(1): 205-20, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16400024

RESUMO

Mouse models for autosomal-dominant polycystic kidney disease (ADPKD), derived from homozygous targeted disruption of Pkd1 gene, generally die in utero or perinatally because of systemic defects. We introduced a loxP site and a loxP-flanked mc1-neo cassette into introns 30 and 34, respectively, of the Pkd1 locus to generate a conditional, targeted mutation. Significantly, before excision of the floxed exons and mc1-neo from the targeted locus by Cre recombinase, mice homozygous for the targeted allele appeared normal at birth but developed polycystic kidney disease with a slower progression than that of Pkd-null mice. Further, the homozygotes continued to produce low levels of full-length Pkd1-encoded protein, suggesting that slight Pkd1 expression is sufficient for renal cyst formation in ADPKD. In this viable model, up-regulation of heparin-binding epidermal growth factor-like growth factor accompanied increased epidermal growth factor receptor signaling, which may be involved in abnormal proliferation of the cyst-lining epithelia. Increased apoptosis in cyst epithelia was only observed in the later period that correlated with the cyst regression. Abnormalities in Na(+)/K(+)-ATPase, aquaporin-2, and vasopressin V2 receptor expression were also identified. This mouse model may be suitable for further studies of progression and therapeutic interventions of ADPKD.


Assuntos
Modelos Animais de Doenças , Doenças Renais Policísticas/genética , Proteínas/genética , Animais , Apoptose/fisiologia , Aquaporina 2/metabolismo , Western Blotting , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Knockout , Mutação , Doenças Renais Policísticas/patologia , Proteínas/metabolismo , Receptores de Vasopressinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPase Trocadora de Sódio-Potássio/metabolismo , Canais de Cátion TRPP
15.
Dev Biol ; 270(2): 513-24, 2004 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-15183730

RESUMO

In developing limb buds, Msx2 transcripts are expressed in the apical ectodermal ridge (AER) and in various regions of the limb mesenchyme. To identify DNA sequences responsible for Msx2 expression in the AER, we characterized the expression of LacZ reporter constructs driven by chicken Msx2 regulatory sequences in transgenic mice. We have identified a 55-bp enhancer that can direct AER-specific reporter gene expression. This 55-bp enhancer contains three elements that are evolutionary conserved among five vertebrate Msx2 genomic sequences. AER expression of reporter constructs in transgenic mice is lost or reduced when mutations are introduced into each of these three regions. Moreover, changing the relative orientation by reverse complementing one of the three elements also results in loss of expression, suggesting that the relative orientations of transcription factor binding is important. To identify the transcription factor(s) binding to these elements, we conducted one-hybrid screening and identified Dlx5 and Sox11. Both Dlx5 and Sox11 are expressed in the AER, and the proteins encoded by these genes bind to separate conserved elements, supporting their possible roles in regulating Msx2 expression.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Ectoderma/metabolismo , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Camundongos/embriologia , Animais , Sequência de Bases , Sequência Conservada/genética , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Elementos Facilitadores Genéticos/genética , Genes Reporter/genética , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Luciferases , Camundongos/metabolismo , Camundongos Transgênicos , Dados de Sequência Molecular , Fatores de Transcrição SOXC , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido
16.
Dev Biol ; 269(1): 109-22, 2004 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-15081361

RESUMO

Several bone morphogenetic proteins (BMPs) are expressed in the apical ectodermal ridge (AER), a critical signaling center that directs the outgrowth and patterning of limb mesoderm, but little is known about their function. To study the functions of apical ectodermal BMPs, an AER-specific promoter element from the Msx2 gene was used to target expression of the potent BMP antagonist noggin to the apical ectoderm of the limbs of transgenic mice. Msx2-noggin mutant mice have severely malformed limbs characterized by syndactyly, postaxial polydactyly, and dorsal transformations of ventral structures indicated by absence of ventral footpads and presence of supernumerary ventral nails. Mutant limb buds exhibit a dorsoventral (DV) and anteroposterior (AP) expansion in the extent of the AER. AER activity persists longer than normal and is maintained in regions of the apical ectoderm where its activity normally ceases. Mutant limbs possess a broad band of mesodermal tissue along the distal periphery that is absent from normal limbs and which fails to undergo the apoptosis that normally occurs in the subectodermal mesoderm. Taken together, our results suggest that apical ectodermal BMPs may delimit the boundaries of the AER by preventing adjacent nonridge ectodermal cells from becoming AER cells; negatively modulate AER activity and thus fine-tune the strength of AER signaling; and regulate the apoptosis of the distal subectodermal mesoderm that occurs as AER activity attenuates, an event that is essential for normal limb development. Our results also confirm that ectodermal BMP signaling regulates DV patterning.


Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Ectoderma/fisiologia , Extremidades/embriologia , Animais , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas de Transporte , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Extremidades/fisiologia , Proteínas de Homeodomínio , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/metabolismo , Camundongos , Mutação , Proteínas/genética , Proteínas/metabolismo
17.
Eur J Neurosci ; 18(7): 1786-96, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14622213

RESUMO

We isolated and characterized a 4.8-kb 5' flanking region of the rat A2A adenosine receptor (A2A-R) gene in the present study. Promoter activity was observed with this DNA fragment in PC12 cells and C6 cells which contain endogenous A2A-Rs. A fusion fragment consisting of the 4.8-kb promoter-proximal DNA fragment of the A2A-R gene, and the coding region of lacZ was utilized to produce mice harbouring the fusion gene. In three independent founder lines, proteins and transcripts of the transgene were found in many areas of the central nervous system (CNS), but not in three peripheral tissues examined. Double immunohistochemical analyses revealed that the transgene was coexpressed with endogenous A2A-R and proper neuronal markers in the brain. Specifically, the transgene in the striatum was found in the enkephalin-containing GABAergic neurons and in the cholinergic neurons as was found for the endogenous A2A-R. However, a selectively enriched striatal expression of the transgene was not found as was observed for the endogenous A2A-R. Collectively, the 4.8-kb promoter-proximal DNA fragment of the rat A2A-R gene contains important element(s) to direct its expression in the CNS where functional A2A-R are found, but were not sufficient to confer the highly concentrated expression of the striatal A2A-R. Furthermore, expressions of A2A-R and the transgene were found in both neurons and astrocytes, suggesting that adenosine might mediate its function through A2A-R in both cell types.


Assuntos
Região 5'-Flanqueadora , Sistema Nervoso Central/metabolismo , Regiões Promotoras Genéticas , Receptor A2A de Adenosina/genética , Adenilil Ciclases/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Southern Blotting , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sistema Nervoso Central/anatomia & histologia , Sistema Nervoso Central/efeitos dos fármacos , Fragmentação do DNA , Expressão Gênica/efeitos dos fármacos , Genes Reporter/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Reação em Cadeia da Polimerase , RNA/metabolismo , Ratos , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/metabolismo , Transfecção , Tubulina (Proteína)/metabolismo , beta-Galactosidase/metabolismo
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