RESUMO
The maintenance of cycling cell lineages relies on undifferentiated subpopulations consisting of stem and progenitor pools. Features that delineate these cell types are undefined for many lineages, including spermatogenesis, which is supported by an undifferentiated spermatogonial population. Here, we generated a transgenic mouse line in which spermatogonial stem cells are marked by expression of an inhibitor of differentiation 4 (Id4)-green fluorescent protein (Gfp) transgene. We found that Id4-Gfp(+) cells exist primarily as a subset of the type A(single) pool, and their frequency is greatest in neonatal development and then decreases in proportion during establishment of the spermatogenic lineage, eventually comprising â¼ 2% of the undifferentiated spermatogonial population in adulthood. RNA sequencing analysis revealed that expression of 11 and 25 genes is unique for the Id4-Gfp(+)/stem cell and Id4-Gfp(-)/progenitor fractions, respectively. Collectively, these findings provide the first definitive evidence that stem cells exist as a rare subset of the A(single) pool and reveal transcriptome features distinguishing stem cell and progenitor states within the mammalian male germline.
Assuntos
Células Germinativas/citologia , Proteínas Inibidoras de Diferenciação/metabolismo , Células-Tronco/citologia , Testículo/citologia , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Inibidoras de Diferenciação/genética , Masculino , Camundongos , Camundongos Transgênicos , Espermatogênese/genética , Espermatogônias/metabolismo , Células-Tronco/metabolismo , Testículo/metabolismo , TranscriptomaRESUMO
The Hox genes pattern the anterior-posterior axis in developing embryos through tightly regulated, partially overlapping, temporal and spatial expression domains. Initial regulation of Hox expression is important to establish these overlapping transcription domains. The Cdx homeodomain factors have been proposed as Hox regulators, but their precise role and mechanism during this regulatory interaction remain unclear. In Xenopus embryos, HoxC8 transcripts begin to accumulate during mid/late gastrula. Cdx1 overexpression and knockdown lead to precocious or slower HoxC8 expression, respectively. The mouse HoxC8 early enhancer when introduced into Xenopus embryos recapitulates the endogenous XHoxC8 temporal expression pattern and shows the same responsiveness to Cdx1 regulation. Three pairs of conserved Cdx binding sites were identified within the HoxC8 early enhancer. We demonstrate that Cdx1 binds directly these responsive elements during embryogenesis, as part of the mechanism for the timely activation of HoxC8 expression. We define the function and mechanism of Cdx1 regulation on HoxC8 expression and suggest the possibility that the temporal changes in Cdx activity levels during gastrulation, combined with differential DNA binding affinity, might play a role in the establishment of Hox sequential activation.
Assuntos
Desenvolvimento Embrionário/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/fisiologia , Animais , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Proteínas de Homeodomínio/genética , Camundongos , Proteínas de Xenopus/biossíntese , Xenopus laevisRESUMO
Hoxb13 is robustly transcribed in derivatives of posterior endoderm including the colon, rectum, and the prostate gland. Transcriptional activity in the prostate persists unabated under conditions of androgen deprivation and throughout the course of disease progression in a mouse prostate cancer model. To elucidate the molecular basis of prostate-restricted transcriptional activation of Hoxb13, a bacterial artificial chromosome (BAC)-based reporter gene deletion analysis was performed in transgenic mice. Two regions downstream of the Hoxb13 coding region were found to be required to support transcriptional activity in the prostate but were completely dispensable for expression in the colon and rectum. Bioinformatic analyses of one region identified a 37-bp element conserved in mammals. This element, which bears two potential binding sites for Forkhead class transcription factors, is occupied by FOXA1 in a human prostate cancer cell line. Precise replacement of this enhancer with an extended LoxP site in the context of a 218,555-bp BAC reporter nearly extinguished Hoxb13-mediated transcriptional activity in the mouse prostate. These data demonstrate that FOXA1 directly regulates HOXB13 in human prostate epithelial cells, and show that this prostate-specific regulatory mechanism is conserved in mice.
Assuntos
Elementos Facilitadores Genéticos , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/metabolismo , Próstata/metabolismo , Animais , Sequência de Bases , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Regulação da Expressão Gênica , Genes Reporter , Fator 3-alfa Nuclear de Hepatócito/genética , Proteínas de Homeodomínio/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Próstata/citologiaRESUMO
Whole Exome Sequencing (WES) is a powerful clinical diagnostic tool for discovering the genetic basis of many diseases. A major shortcoming of WES is uneven coverage of sequence reads over the exome targets contributing to many low coverage regions, which hinders accurate variant calling. In this study, we devised two novel metrics, Cohort Coverage Sparseness (CCS) and Unevenness (UE) Scores for a detailed assessment of the distribution of coverage of sequence reads. Employing these metrics we revealed non-uniformity of coverage and low coverage regions in the WES data generated by three different platforms. This non-uniformity of coverage is both local (coverage of a given exon across different platforms) and global (coverage of all exons across the genome in the given platform). The low coverage regions encompassing functionally important genes were often associated with high GC content, repeat elements and segmental duplications. While a majority of the problems associated with WES are due to the limitations of the capture methods, further refinements in WES technologies have the potential to enhance its clinical applications.
Assuntos
Mineração de Dados/métodos , Sequenciamento do Exoma/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , HumanosRESUMO
Fibroblast growth factor receptor (FGFR) gene products (Fgfr1, Fgfr2, Fgfr3) are widely expressed by embryonic neural progenitor cells throughout the CNS, yet their functional role in cerebral cortical development is still unclear. To understand whether the FGF pathways play a role in cortical development, we attenuated FGFR signaling by expressing a tyrosine kinase domain-deficient Fgfr1 (tFgfr1) gene construct during embryonic brain development. Mice carrying the tFgfr1 transgene under the control of the Otx1 gene promoter have decreased thickness of the cerebral cortex in frontal and temporal areas because of decreased number of pyramidal neurons and disorganization of pyramidal cell dendritic architecture. These alterations may be, in part, attributable to decreased genesis of T-Brain-1-positive early glutamatergic neurons and, in part, to a failure to maintain radial glia fibers in medial prefrontal and temporal areas of the cortical plate. No changes were detected in cortical GABAergic interneurons, including Cajal-Retzius cells or in the basal ganglia. Behaviorally, tFgfr1 transgenic mice displayed spontaneous and persistent locomotor hyperactivity that apparently was not attributable to alterations in subcortical monoaminergic systems, because transgenic animals responded to both amphetamine and guanfacine, an alpha2A adrenergic receptor agonist. We conclude that FGF tyrosine kinase signaling may be required for the genesis and growth of pyramidal neurons in frontal and temporal cortical areas, and that alterations in cortical development attributable to disrupted FGF signaling are critical for the inhibitory regulation of motor behavior.
Assuntos
Lobo Frontal/metabolismo , Hipercinese/genética , Malformações do Sistema Nervoso/genética , Células Piramidais/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Lobo Temporal/metabolismo , Agonistas alfa-Adrenérgicos/farmacologia , Anfetamina , Animais , Diferenciação Celular/genética , Divisão Celular , Lobo Frontal/anormalidades , Lobo Frontal/patologia , Ácido Glutâmico/metabolismo , Guanfacina/farmacologia , Humanos , Hipercinese/induzido quimicamente , Hipercinese/tratamento farmacológico , Camundongos , Camundongos Transgênicos , Malformações do Sistema Nervoso/metabolismo , Malformações do Sistema Nervoso/patologia , Inibição Neural/genética , Células Piramidais/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Transdução de Sinais/genética , Transtorno de Movimento Estereotipado/induzido quimicamente , Transtorno de Movimento Estereotipado/tratamento farmacológico , Transtorno de Movimento Estereotipado/genética , Lobo Temporal/anormalidades , Lobo Temporal/patologiaRESUMO
Transgenic mouse models are valuable resources for analyzing functions of genes involved in human diseases. Mouse models provide critical insights into biological processes, including in vivo visualization of vasculature critical to our understanding of the immune system. Generating transgenic mice requires the capture and modification of large-insert DNAs representing genes of interest. We have developed a methodology using a yeast-bacterial shuttle vector, pClasper, that enables the capture and modification of bacterial artificial chromosomes (BAC)-sized DNA inserts. Numerous improvements and technical advances in the original pClasper vector have allowed greater flexibility and utility in this system. Examples of such pClasper mediated gene modifications include: Claspette-mediated capture of large-insert genomic fragments from BACs-human polycystic kidney disease-1 (PKD1); modification of pClasperA clones by the RareGap method-PKD1 mutations; Claspette-mediated modification of pClasper clones-mouse albumin-1 gene; and, of most relevance to our interest in lymph node vasculature-Claspimer-mediated modification of pClasper clones-high endothelial venule and lymphatic vessel genes. Mice that have been generated with these methods include mice with fluorescent high endothelial venules.
Assuntos
Doenças Renais Policísticas/genética , Recombinação Genética , Saccharomyces cerevisiae/genética , Animais , Cromossomos Artificiais Bacterianos , Vetores Genéticos , Humanos , Camundongos , Camundongos TransgênicosRESUMO
The caudal family of transcription factors specifies posterior structures during mouse development. We describe the cis-regulatory regions that control mouse Cdx2 expression in the posterior neural tube and mesoderm. An 11.4 kb genomic fragment directs reporter gene expression in a pattern reflecting endogenous Cdx2 expression. A crucial enhancer is located in a 1 kb fragment upstream of the Cdx2 transcriptional start site. This enhancer by itself directs reporter gene expression to more anterior levels in the neural tube compared to the endogenous Cdx2 expression, suggesting the presence of negative regulatory elements outside the 1 kb fragment. A second enhancer, located in the first intron directs robust expression to the posterior two-thirds of the developing embryo in a pattern that is ectopic to Cdx2 expression. The intronic enhancer activity is silenced in the context of the larger 11.4 kb reporter construct. Intron 1 contains two independent enhancers that specifically direct expression to mesoderm (MSE) and neural tube (NSE). Phylogenetic comparison of vertebrate Cdx2 sequences indicates several conserved regions of sequences within the three-enhancer regions. A transcription factor database search suggests potential binding sites for factors involved in FGF and Wnt signaling pathways.
Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Filogenia , Fatores de Transcrição/genética , Animais , Pareamento de Bases , Sequência de Bases , Fator de Transcrição CDX2 , Componentes do Gene , Perfilação da Expressão Gênica , Genes Reporter/genética , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de Transcrição/metabolismoRESUMO
The Hoxc8 early enhancer that controls the initiation and establishment of Hoxc8 expression in the developing mouse embryo is found in different vertebrate lineages including mammals, birds and fish. Mouse and Fugu Hoxc8 early enhancers (200 bp) have diverged in the composition of elements located towards the 3' region. However, they share cis-acting elements A-E located in the 5' region. Mutations at these elements in the context of the mouse Hoxc8 early enhancer affect reporter gene expression in the posterior neural tube, somites and lateral plate mesoderm of day 9.5 mouse embryos. Here, we demonstrate that mutations introduced at the same elements but in the context of the Fugu Hoxc8 early enhancer had different consequences on the reporter gene expression in transgenic mouse embryos. Furthermore, in contrast to the mouse enhancer the Fugu enhancer does not utilize elements D and E in achieving posterior neural tube and somite expression. These results suggest that the diverged sequences prevent regulatory interactions at conserved cis-acting elements. We propose that divergent sequences modify regulatory interactions at conserved elements by providing a "contextual change". Our finding that the enhancer elements do not act in a unitary fashion but function in the context of the surrounding sequence brings a new dimension to the study of cis-regulatory evolution.
Assuntos
Elementos Facilitadores Genéticos/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Variação Genética , Proteínas de Homeodomínio/genética , Animais , Sequência de Bases , Análise Mutacional de DNA , Embrião de Mamíferos/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Perfilação da Expressão Gênica , Genes Reporter/genética , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação/genética , Tubo Neural/metabolismo , Somitos/metabolismo , TakifuguRESUMO
Gene transfer technologies in mammals are the focus of renewed interest owing to the recent emphasis on analyzing gene function in the postgenomic era. Three important developments in this area include transgenics, gene targeting and nuclear transfer or animal cloning. These technological innovations have enhanced our ability to analyze gene function at the level of the whole organism and have provided the means to modify gene expression. This review discusses the origins and current status of transgenic technologies. Various applications and technologies including chromosome engineering, stem cells, gene traps and modification of livestock are presented. The impact of mouse technologies and genomics on functional analyses is also discussed.
Assuntos
Animais Geneticamente Modificados/genética , Genômica , Animais , Biotecnologia , Clonagem Molecular , Clonagem de Organismos , Biologia Computacional , Modelos Animais de Doenças , Expressão Gênica , Marcação de Genes , Genes Reguladores , Teste de Complementação Genética , Terapia Genética , Humanos , Camundongos , Células-Tronco/metabolismoRESUMO
The non-coding intergenic regions of Hox genes are remarkably conserved among mammals. To determine the usefulness of this sequence for phylogenetic comparisons, we sequenced an 800-bp fragment of the Hoxc9-Hoxc8 intergenic region from several species belonging to different mammalian clades. Results obtained from the phylogenetic analysis are congruent with currently accepted mammalian phylogeny. Additionally, we found a TC mini satellite repeat polymorphism unique to felines. This polymorphism may serve as a useful marker to differentiate between mammalian species or as a genetic marker in feline matings. This study demonstrates usefulness of a comparative approach employing non-coding regions of Hox gene complexes.
Assuntos
DNA Intergênico , Proteínas de Homeodomínio/genética , Mamíferos/genética , Filogenia , Animais , Elementos Facilitadores Genéticos , Evolução Molecular , Variação Genética , Humanos , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
The Hoxc8 early enhancer is a 200 bp region that controls the early phase of Hoxc8 expression during mouse embryonic development. This enhancer defines the domain of Hoxc8 expression in the neural tube and mesoderm of the posterior regions of the developing embryo. Five distinct cis-acting elements, A-E, were previously shown to govern early phase Hoxc8 expression. Significant divergence between mammalian and fish Hoxc8 early enhancer sequences and activities suggested additional cis-acting elements. Here we describe four additional cis-acting elements (F-I) within the 200 bp Hoxc8 early enhancer region identified by comparative regulatory analysis and transgene-mutation studies. These elements affect posterior neural tube and mesoderm expression of the reporter gene, either singly or in combination. Surprisingly, these new elements are missing from the zebrafish and Fugu Hoxc8 early enhancer sequences. Considering that fish enhancers direct robust reporter expression in transgenic mouse embryos, it is tempting to postulate that fish and mammalian Hoxc8 early enhancers utilize different sets of elements to direct Hoxc8 early expression. These observations reveal a remarkable plasticity in the Hoxc8 early enhancer, suggesting different modes of initiation and establishment of Hoxc8 expression in different species. We postulate that extensive restructuring and remodeling of Hox cis-regulatory regions occurring in different taxa lead to relatively different Hox expression patterns, which in turn may act as a driving force in generating diverse axial morphologies.
Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Camundongos/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Sequência de Bases , Clonagem Molecular , Análise Mutacional de DNA , Primers do DNA , Genes Reporter/genética , Mesoderma/metabolismo , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
The Dlx genes are involved in early vertebrate morphogenesis, notably of the head. The six Dlx genes of mammals are arranged in three convergently transcribed bigene clusters. In this study, we examine the regulation of the Dlx3-7 cluster of the mouse. We obtained and sequenced human and mouse P1 clones covering the entire Dlx3-7 cluster. Comparative analysis of the human and mouse sequences revealed several highly conserved noncoding regions within 30 kb of the Dlx3-7-coding regions. These conserved elements were located both 5' of the coding exons of each gene and in the intergenic region 3' of the exons, suggesting that some enhancers might be shared between genes. We also found that the protein sequence of Dlx7 is evolving more rapidly than that of Dlx3. We conducted a functional study of the 79-kb mouse genomic clone to locate cis-element activity able to reproduce the endogenous expression pattern by using transgenic mice. We inserted a lacZ reporter gene into the first exon of the Dlx3 gene by using homologous recombination in yeast. Strong lacZ expression in embryonic (E) stage E9.5 and E10.5 mouse embryos was found in the limb buds and first and second visceral arches, consistent with the endogenous Dlx3 expression pattern. This result shows that the 79-kb region contains the major cis-elements required to direct the endogenous expression of Dlx3 at stage E10.5. To test for enhancer location, we divided the construct in the mid-intergenic region and injected the Dlx3 gene portion. This shortened fragment lacking Dlx7-flanking sequences is able to drive expression in the limb buds but not in the visceral arches. This observation is consistent with a cis-regulatory enhancer-sharing model within the Dlx bigene cluster.
Assuntos
Proteínas de Homeodomínio/genética , Família Multigênica , Fatores de Transcrição/genética , Animais , Sequência de Bases , Sequência Conservada , DNA/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Genoma Humano , Humanos , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
There is considerable interest in understanding how cis-regulatory modifications drive morphological changes across species. Because developmental regulatory genes, including Hox genes, are remarkably conserved, their noncoding regulatory regions are likely sources for variations. Modifications of Hox cis-regulatory elements have potential to alter Hox gene expression and, hence, axial morphologies. In vertebrates, differences in the axial levels of Hox gene expression correlate with differences in the number and relative position of thoracic vertebrae. Variation in cis-regulatory elements of Hox genes can be identified by comparative sequence and reporter gene analyses in transgenic mouse embryos. Using these approaches, we show a remarkable divergence of the Hoxc8 early enhancers between mammals and fishes representing diverse axial morphologies. Extensive restructuring of the Hoxc8 early enhancer including nucleotide substitutions, inversion, and divergence result in distinct patterns of reporter gene expression along the embryonic axis. Our results provide an evolutionary perspective on how the enhancer elements are engineered and support the hypothesis that remodeling of Hox regulatory elements in different species has played a significant role in generating morphological diversity.