RESUMO
Development of mammalian lung involves reiterative outgrowth and branching of an epithelial tube into the surrounding mesenchymal bed. Each coordinated growth and branching cycle is driven by reciprocal signalling between epithelial and adjacent mesenchymal cells. This signalling network includes FGF, SHH, BMP4 and other pathways. We have characterized lung defects in 36Pub mice carrying a deletion that removes an antagonist of FGF signalling, Spry2. Spry2 deficient mice show an enlarged cystic structure located in the terminus of each lobes. Our study shows that Spry2 deficient lungs have reduced lung branching and the cystic structure forms in the early lung development stage. Furthermore, mice carrying a targeted disruption of Spry2 fail to complement the lung phenotype characterized in 36Pub mice. A Spry2-BAC transgene rescues the defect. Interestingly, cystic structure growth is accompanied by the reduced and spatially disorganized expression of Fgf10 and elevated expression of Shh and Bmp4. Altered signalling balance due to the loss of Spry2 causes a delayed branch cycle and cystic growth. Our data underscores the importance of restricting cellular responsiveness to signalling and highlights the interplay between morphogenesis events and spatial localization of gene expression.
Assuntos
Embrião de Mamíferos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Pulmão/metabolismo , Proteínas de Membrana/genética , Transdução de Sinais/genética , Animais , Proteína Morfogenética Óssea 4/genética , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/patologia , Fator 10 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Hibridização In Situ , Pulmão/embriologia , Pulmão/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Morfogênese/genética , Proteínas Serina-Treonina Quinases , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Specific mammalian genes functionally and dynamically associate together within the nucleus. Yet, how an array of many genes along the chromosome sequence can be spatially organized and folded together is unknown. We investigated the 3D structure of a well-annotated, highly conserved 4.3-Mb region on mouse chromosome 14 that contains four clusters of genes separated by gene "deserts." In nuclei, this region forms multiple, nonrandom "higher order" structures. These structures are based on the gene distribution pattern in primary sequence and are marked by preferential associations among multiple gene clusters. Associating gene clusters represent expressed chromatin, but their aggregation is not simply dependent on ongoing transcription. In chromosomes with aggregated gene clusters, gene deserts preferentially align with the nuclear periphery, providing evidence for chromosomal region architecture by specific associations with functional nuclear domains. Together, these data suggest dynamic, probabilistic 3D folding states for a contiguous megabase-scale chromosomal region, supporting the diverse activities of multiple genes and their conserved primary sequence organization.
Assuntos
Mapeamento Cromossômico , Cromossomos de Mamíferos/química , Cromossomos de Mamíferos/genética , Genoma , Animais , Núcleo Celular/genética , Células Cultivadas , Cromatina/genética , Fibroblastos/metabolismo , Hibridização in Situ Fluorescente , Camundongos , Modelos Biológicos , Células NIH 3T3RESUMO
Evolution of facial morphology arises from variation in the activity of developmental regulatory networks that guide the formation of specific craniofacial elements. Importantly, the acquisition of novel morphology must be integrated with a phylogenetically inherited developmental program. We have identified a unique region of the secondary palate associated with the periodic formation of rugae during the rostral outgrowth of the face. Rugae function as SHH signaling centers to pattern the elongating palatal shelves. We have found that a network of signaling genes and transcription factors is spatially organized relative to palatal rugae. Additionally, the first formed ruga is strategically positioned at the presumptive junction of the future hard and soft palate that defines anterior-posterior differences in regional growth, mesenchymal gene expression, and cell fate. We propose a molecular circuit integrating FGF and BMP signaling to control proliferation and differentiation during the sequential formation of rugae and inter-rugae domains in the palatal epithelium. The loss of p63 and Sostdc1 expression and failed rugae differentiation highlight that coordinated epithelial-mesenchymal signaling is lost in the Fgf10 mutant palate. Our results establish a genetic program that reiteratively organizes signaling domains to coordinate the growth of the secondary palate with the elongating midfacial complex.
Assuntos
Fator 10 de Crescimento de Fibroblastos/genética , Proteínas Hedgehog/genética , Palato/metabolismo , Transdução de Sinais/genética , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/fisiologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Epitélio/metabolismo , Feminino , Fator 10 de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Proteínas Hedgehog/fisiologia , Hibridização In Situ , Masculino , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Mutação , Palato/embriologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Fatores de TempoRESUMO
Walleye (Sander vitreus) is an important game fish throughout much of North America. We evaluated the performance of the Wisconsin bioenergetics model for walleye in the laboratory. Walleyes were fed rainbow smelt (Osmerus mordax) in four laboratory tanks during a 126-day experiment. Based on a statistical comparison of bioenergetics model predictions of monthly consumption with the observed monthly consumption, we concluded that the bioenergetics model significantly underestimated food consumption by walleye in the laboratory. The degree of underestimation appeared to depend on the feeding rate. For the tank with the lowest feeding rate (1.4% of walleye body weight per day), the agreement between the bioenergetics model prediction of cumulative consumption over the entire 126-day experiment and the observed cumulative consumption was remarkably close, as the prediction was within 0.1% of the observed cumulative consumption. Feeding rates in the other three tanks ranged from 1.6% to 1.7% of walleye body weight per day, and bioenergetics model predictions of cumulative consumption over the 126-day experiment ranged between 11 and 15% less than the observed cumulative consumption.
Assuntos
Metabolismo Energético , Modelos Biológicos , Perciformes/metabolismo , Animais , Ingestão de Alimentos/fisiologiaRESUMO
The piebald deletion complex is a set of overlapping chromosomal deficiencies on distal mouse chromosome 14. We surveyed the functional genetic content of the piebald deletion region in an essential gene mutagenesis screen of 952 genomes to recover seven lethal mutants. The ENU-induced mutations were mapped to define genetic intervals using the piebald deletion panel. Lethal mutations included loci required for establishment of the left-right embryonic axis and a loss-of-function allele of Phr1 resulting in respiratory distress at birth. A functional map of the piebald region integrates experimental genetic data from the deletion panel, mutagenesis screen, and the targeted disruption of specific genes. A comparison of several genomic intervals targeted in regional mutagenesis screens suggests that the piebald region is characterized by a low gene density and high essential gene density with a distinct genomic content and organization that supports complex regulatory interactions and promotes evolutionary stability.
Assuntos
Proteínas de Transporte/genética , Deleção Cromossômica , Cromossomos de Mamíferos/genética , Mutagênese/genética , Alquilantes/toxicidade , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sequência de Bases , Mapeamento Cromossômico , Análise Mutacional de DNA , Etilnitrosoureia/toxicidade , Feminino , Genes Essenciais , Genes Letais , Genótipo , Pneumopatias/genética , Pneumopatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutagênese/efeitos dos fármacos , Fenótipo , Ubiquitina-Proteína LigasesRESUMO
AIM: The aim of this study is to measure the seroprevalence of cytomegalovirus (CMV) infection in 3.5-year-old children, and identify the determinants of seropositivity. METHODS: A total of 1714 children were enrolled at birth. Approximately half were small for gestational age and half were appropriate for gestational age. Information on the children was collected at birth, 1 year and 3.5 years. At 3.5 years blood was collected and tested for CMV-specific immunoglobulin by an enzyme-linked immunosorbent assay in 530 children. RESULTS: The weighted seroprevalence of CMV was 32.8% (95% confidence interval (CI) 27.4-38.1%). The seroprevalence of CMV varied markedly by ethnicity (European: 26.5% (95% CI 20.9-32.2%); Maori: 68.0% (44.0-92.0%); Pacific: 74.5% (56.3-92.6%); Indian: 50.0% (20.2-79.8%); Chinese: 47.2% (10.8-83.5%); Other: 21.9% (0.0-52.7%); P < 0.001). Socio-economic factors, number of siblings, day care centres attendance, maternal smoking, breastfeeding and other factors examined were not related to CMV seropositivity. CONCLUSIONS: The seroprevalence of CMV in New Zealand pre-school children is similar to that reported from other developed countries. The finding of marked ethnic differences is unexplained by socio-economic factors, or other factors that were examined.
Assuntos
Infecções por Citomegalovirus/epidemiologia , Citomegalovirus/isolamento & purificação , Estudos de Casos e Controles , Pré-Escolar , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/etnologia , Características da Família , Feminino , Humanos , Lactente , Recém-Nascido , Recém-Nascido Pequeno para a Idade Gestacional/sangue , Masculino , Nova Zelândia/epidemiologia , Fatores de Risco , Estudos SoroepidemiológicosRESUMO
The formation of the palate involves the coordinated outgrowth, elevation and midline fusion of bilateral shelves leading to the separation of the oral and nasal cavities. Reciprocal signaling between adjacent fields of epithelial and mesenchymal cells directs palatal shelf growth and morphogenesis. Loss of function mutations in genes encoding FGF ligands and receptors have demonstrated a critical role for FGF signaling in mediating these epithelial-mesenchymal interactions. The Sprouty family of genes encode modulators of FGF signaling. We have established that mice carrying a deletion that removes the FGF signaling antagonist Spry2 have cleft palate. We show that excessive cell proliferation in the Spry2-deficient palate is accompanied by the abnormal progression of shape changes and movements required for medially directed shelf outgrowth and midline contact. Expression of the FGF responsive transcription factors Etv5, Msx1, and Barx1, as well as the morphogen Shh, is restricted to specific regions of the developing palate. We detected elevated and ectopic expression of these transcription factors and disorganized Shh expression in the Spry2-deficient palate. Mice carrying a targeted disruption of Spry2 fail to complement the craniofacial phenotype characterized in Spry2 deletion mice. Furthermore, a Spry2-BAC transgene rescues the palate defect. However, the BAC transgenic mouse lines express reduced levels of Spry2. The resulting hypomorphic phenotype demonstrates that palate development is Spry2 dosage sensitive. Our results demonstrate the importance of proper FGF signaling thresholds in regulation of epithelial-mesenchymal interactions and cellular responses necessary for coordinated morphogenesis of the face and palate.
Assuntos
Padronização Corporal/genética , Dosagem de Genes/fisiologia , Proteínas de Membrana/fisiologia , Palato/embriologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Diferenciação Celular/genética , Movimento Celular/genética , Fissura Palatina/genética , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/fisiologia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/biossíntese , Proteínas Hedgehog/deficiência , Proteínas Hedgehog/genética , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases , Transdução de Sinais/genéticaRESUMO
Dlx3, a homeodomain transcription factor, is essential for placental development in the mouse. The Dlx3(-/-) mouse embryo dies at embryonic d 9.5-10 putatively due to placental failure. To develop a more comprehensive understanding of the gene profile regulated by Dlx3, microarray analysis was used to determine differences in gene expression within the placenta of Dlx3(+/+) and Dlx3(-/-) mice. Array analysis revealed differential expression of 401 genes, 33 genes in which signal to log ratio values of null/wild-type were lower than -0.5 or higher than 0.5. To corroborate these findings, quantitative real-time PCR was used to confirm differential expression for 11 genes, nine of which displayed reduced expression and two with enhanced expression in the Dlx3(-/-) mouse. Loss of Dlx3 resulted in a marked reduction (>60%) in mRNA expression of placental growth factor (Pgf), a member of the vascular endothelial growth factor family. Consistent with these results, Pgf secretion from placental explants tended to be reduced in the Dlx3(-/-) mice, compared with wild type. To investigate mechanisms of Dlx3 regulation of Pgf gene transcription, we cloned 5.2 kb of the Pgf 5' flanking sequence for use in reporter gene assays. Expression of the Pgf promoter luciferase reporter containing at least three Dlx3 binding sites was increased markedly by overexpression of Dlx3 supporting the conclusion that Dlx3 may have a direct effect on Pgf promoter activity. These studies provide a novel view of the transcriptome regulated by Dlx3 in mouse placenta. Dlx3 is specifically required for full expression and secretion of Pgf in vivo. Moreover, in vitro studies support the conclusion that Dlx3 is sufficient to directly modulate expression of the Pgf gene promoter in placental cells.
Assuntos
Perfilação da Expressão Gênica , Proteínas de Homeodomínio/fisiologia , Placenta/metabolismo , Prenhez , Fatores de Transcrição/fisiologia , Animais , Coriocarcinoma/genética , Coriocarcinoma/metabolismo , Embrião de Mamíferos , Feminino , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fator de Crescimento Placentário , Placentação , Gravidez , Proteínas da Gravidez/genética , Proteínas da Gravidez/fisiologia , Prenhez/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismoRESUMO
Genetic studies using a set of overlapping deletions centered at the piebald locus on distal mouse chromosome 14 have defined a genomic region associated with respiratory distress and lethality at birth. We have isolated and characterized the candidate gene Phr1 that is located within the respiratory distress critical genomic interval. Phr1 is the ortholog of the human Protein Associated with Myc as well as Drosophila highwire and Caenorhabditis elegans regulator of presynaptic morphology 1. Phr1 is expressed in the embryonic and postnatal nervous system. In mice lacking Phr1, the phrenic nerve failed to completely innervate the diaphragm. In addition, nerve terminal morphology was severely disrupted, comparable with the synaptic defects seen in the Drosophila hiw and C. elegans rpm-1 mutants. Although intercostal muscles were completely innervated, they also showed dysmorphic nerve terminals. In addition, sensory neuron terminals in the diaphragm were abnormal. The neuromuscular junctions showed excessive sprouting of nerve terminals, consistent with inadequate presynaptic stimulation of the muscle. On the basis of the abnormal neuronal morphology seen in mice, Drosophila, and C. elegans, we propose that Phr1 plays a conserved role in synaptic development and is a candidate gene for respiratory distress and ventilatory disorders that arise from defective neuronal control of breathing.
Assuntos
Proteínas de Membrana/genética , Insuficiência Respiratória/genética , Sinapses/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Sequência Conservada , Embrião de Mamíferos/metabolismo , Evolução Molecular , Imunofluorescência , Proteínas de Membrana/metabolismo , Camundongos , Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Piebaldismo/genética , Insuficiência Respiratória/metabolismo , Deleção de Sequência , Sinapses/metabolismoRESUMO
The study of genetic variation in mice offers a powerful experimental platform for understanding gene function. Complex trait analysis, gene-targeting and gene-trapping technologies, as well as insertional and chemical mutagenesis approaches are becoming increasingly sophisticated and provide a variety of options for cataloguing gene activities and interactions. In this review we discuss fundamental and practical concepts related to chemical mutagenesis and we highlight the growing list of strategies for performing mutagenesis screens in mice. Gene-driven and diverse types of phenotype-driven screens provide several options for the recovery of the invaluable variety of alleles generated by chemical mutagenesis. The unique advantages offered using chemical mutagenesis compare favourably to and complement the spectrum of approaches available for functional annotation of the mammalian genome.
Assuntos
Camundongos/genética , Biologia Molecular/métodos , Biologia Molecular/tendências , Mutagênese , AnimaisRESUMO
Mutagenesis screens to isolate a variety of alleles leading to null and non-null phenotypes represent an important approach for the characterization of gene function. Genetic schemes that use visible markers permit the efficient recovery of chemically induced mutations. We have developed a universal reporter system to visibly mark chromosomes for genetic screens in the mouse. The dual-color reporter is based on a single vector that drives the ubiquitous coexpression of the enhanced GFP (EGFP) spectral variants yellow and cyan. We show that widespread expression of the dual-color reporter is readily detected in embryonic stem cells, mice, and throughout developmental stages. CRE-loxP- and FLPe-FRT-mediated deletion of each color cassette demonstrates the modular design of the marker system. Random integration followed by plasmid rescue and sequence-based mapping was used to introduce the marker to a defined genomic location. Thus, single-step placement will simplify the construction of a genomewide bank of marked chromosomes. The dual-color nature of the marker permits complete identification of genetic classes of progeny as embryos or mice in classic regionally directed screens. The design also allows for more efficient and novel schemes, such as marked suppressor screens, in the mouse. The result is a versatile reporter that can be used independently or in combination with the growing sets of deletion and inversion resources to enhance the design and application of a wide variety of genetic schemes for the functional dissection of the mammalian genome.
Assuntos
Genes Reporter , Proteínas Luminescentes/genética , Mutação , Animais , Sequência de Bases , Deleção Cromossômica , Inversão Cromossômica , DNA Recombinante/genética , Marcadores Genéticos , Proteínas de Fluorescência Verde , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Proteínas Recombinantes/genética , Supressão GenéticaRESUMO
The electroconvulsive threshold (ECT) test has been used extensively to determine the protection conferred by antiepileptic drug candidates against induced seizures in rodents. Despite its clinical relevance, the potential of ECT to identify mouse epilepsy models in genetic studies has not been thoroughly assessed. We adopted the ECT test to screen the progeny of ethylnitrosourea treated male C57BL/6J mice. In a small-scale screen, several mutant lines conferring a low threshold to ECT minimal clonic seizures were mapped to the telomeric region of mouse chromosome 2 in independent founder families. This high incidence was suggestive of a single spontaneous event that pre-existed in the founders of mutagenized stock. Genetic and physical mapping led to the discovery that several lines shared a single mutation, Szt1 (seizure threshold-1), consisting of a 300 kb deletion of genomic DNA involving three known genes. Two of these genes, Kcnq2 and Chrna4, are known to be mutated in human epilepsy families. Szt1 homozygotes and heterozygotes display similar phenotypes to those found in the respective Kcnq2 knockout mutant mice, suggesting that Kcnq2 haploinsufficiency is at the root of the Szt1 seizure sensitivity. Our results provide a novel genetic model for epilepsy research and demonstrate that the approach of using ECT to study seizures in mice has the potential to lead to the identification of human epilepsy susceptibility genes.
Assuntos
Epilepsia/genética , Deleção de Sequência , Animais , Sequência de Bases , Constituição Corporal/genética , Mapeamento Cromossômico , Heterozigoto , Hipocampo/metabolismo , Canal de Potássio KCNQ2 , Camundongos , Canais de Potássio/genética , Canais de Potássio/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da MembranaRESUMO
Several developmentally important genomic regions map within the piebald deletion complex on distal mouse chromosome 14. We have combined computational gene prediction and comparative sequence analysis to characterize an approximately 4.3-Mb segment of the piebald region to identify candidate genes for the phenotypes presented by homozygous deletion mice. As a result we have ordered 13 deletion breakpoints, integrated the sequence with markers from a bacterial artificial chromosome (BAC) physical map, and identified 16 known or predicted genes and >1500 conserved sequence elements (CSEs) across the region. The candidate genes identified include Phr1 (formerly Pam) and Spry2, which are mouse homologs of genes required for development in Drosophila melanogaster. Gene content, order, and position are highly conserved between mouse chromosome 14 and the orthologous region of human chromosome 13. Our studies combining computational gene prediction with genetic and comparative genomic analyses provide insight regarding the functional composition and organization of this defined chromosomal region.
Assuntos
Mapeamento Cromossômico , Deleção de Genes , Piebaldismo/genética , Animais , Camundongos , Proteínas do Tecido Nervoso/genética , Receptor de Endotelina B , Receptores de Endotelina/genética , Análise de Sequência de DNARESUMO
Generation of left-right asymmetry is an integral part of the establishment of the vertebrate body plan. Here we show that the Notch signaling pathway plays a primary role in the establishment of left-right asymmetry in mice by directly regulating expression of the Nodal gene. Embryos mutant for the Notch ligand Dll1 or doubly mutant for the Notch1 and Notch2 receptors exhibit multiple defects in left-right asymmetry. Analysis of the enhancer regulating node-specific Nodal expression revealed the presence of binding sites for the RBP-J protein, the primary transcriptional mediator of Notch signaling. Mutation of these sites destroyed the ability of this enhancer to direct node-specific gene expression in transgenic mice. Our results demonstrate that Dll1-mediated Notch signaling is essential for generation of left-right asymmetry, and that the Notch pathway acts upstream of Nodal expression during left-right asymmetry determination in mice.
Assuntos
Padronização Corporal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Membrana/fisiologia , Fator de Crescimento Transformador beta/genética , Animais , Sítios de Ligação , Cílios/genética , Cílios/metabolismo , Elementos Facilitadores Genéticos , Peptídeos e Proteínas de Sinalização Intracelular , Fatores de Determinação Direita-Esquerda , Proteínas de Membrana/metabolismo , Camundongos , Mutação , Proteína Nodal , Receptores Notch , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Biophysical, chemical, and nanoscience approaches to the study of nuclear structure and activity have been developing recently and hold considerable promise. A selection of fundamental problems in genome organization and function are reviewed and discussed in the context of these new perspectives and approaches. Advancing these concepts will require coordinated networks of physicists, chemists, and materials scientists collaborating with cell, developmental, and genome biologists.