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Xenbase (https://www.xenbase.org/), the Xenopus model organism knowledgebase, is a web-accessible resource that integrates the diverse genomic and biological data from research on the laboratory frogs Xenopus laevis and Xenopus tropicalis. The goal of Xenbase is to accelerate discovery and empower Xenopus research, to enhance the impact of Xenopus research data, and to facilitate the dissemination of these data. Xenbase also enhances the value of Xenopus data through high-quality curation, data integration, providing bioinformatics tools optimized for Xenopus experiments, and linking Xenopus data to human data, and other model organisms. Xenbase also plays an indispensable role in making Xenopus data interoperable and accessible to the broader biomedical community in accordance with FAIR principles. Xenbase provides annotated data updates to organizations such as NCBI, UniProtKB, Ensembl, the Gene Ontology consortium, and most recently, the Alliance of Genomic Resources, a common clearing house for data from humans and model organisms. This article provides a brief overview of key and recently added features of Xenbase. New features include processing of Xenopus high-throughput sequencing data from the NCBI Gene Expression Omnibus; curation of anatomical, physiological, and expression phenotypes with the newly created Xenopus Phenotype Ontology; Xenopus Gene Ontology annotations; new anatomical drawings of the Normal Table of Xenopus development; and integration of the latest Xenopus laevis v10.1 genome annotations. Finally, we highlight areas for future development at Xenbase as we continue to support the Xenopus research community.
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Bases de Dados Genéticas , Genômica , Animais , Humanos , Xenopus laevis/genética , Xenopus/genética , Biologia ComputacionalRESUMO
BACKGROUND: Ontologies of precisely defined, controlled vocabularies are essential to curate the results of biological experiments such that the data are machine searchable, can be computationally analyzed, and are interoperable across the biomedical research continuum. There is also an increasing need for methods to interrelate phenotypic data easily and accurately from experiments in animal models with human development and disease. RESULTS: Here we present the Xenopus phenotype ontology (XPO) to annotate phenotypic data from experiments in Xenopus, one of the major vertebrate model organisms used to study gene function in development and disease. The XPO implements design patterns from the Unified Phenotype Ontology (uPheno), and the principles outlined by the Open Biological and Biomedical Ontologies (OBO Foundry) to maximize interoperability with other species and facilitate ongoing ontology management. Constructed in Web Ontology Language (OWL) the XPO combines the existing uPheno library of ontology design patterns with additional terms from the Xenopus Anatomy Ontology (XAO), the Phenotype and Trait Ontology (PATO) and the Gene Ontology (GO). The integration of these different ontologies into the XPO enables rich phenotypic curation, whilst the uPheno bridging axioms allows phenotypic data from Xenopus experiments to be related to phenotype data from other model organisms and human disease. Moreover, the simple post-composed uPheno design patterns facilitate ongoing XPO development as the generation of new terms and classes of terms can be substantially automated. CONCLUSIONS: The XPO serves as an example of current best practices to help overcome many of the inherent challenges in harmonizing phenotype data between different species. The XPO currently consists of approximately 22,000 terms and is being used to curate phenotypes by Xenbase, the Xenopus Model Organism Knowledgebase, forming a standardized corpus of genotype-phenotype data that can be directly related to other uPheno compliant resources.
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Ontologias Biológicas , Animais , Ontologia Genética , Humanos , Fenótipo , Xenopus laevisRESUMO
At a fundamental level most genes, signaling pathways, biological functions and organ systems are highly conserved between man and all vertebrate species. Leveraging this conservation, researchers are increasingly using the experimental advantages of the amphibian Xenopus to model human disease. The online Xenopus resource, Xenbase, enables human disease modeling by curating the Xenopus literature published in PubMed and integrating these Xenopus data with orthologous human genes, anatomy, and more recently with links to the Online Mendelian Inheritance in Man resource (OMIM) and the Human Disease Ontology (DO). Here we review how Xenbase supports disease modeling and report on a meta-analysis of the published Xenopus research providing an overview of the different types of diseases being modeled in Xenopus and the variety of experimental approaches being used. Text mining of over 50,000 Xenopus research articles imported into Xenbase from PubMed identified approximately 1,000 putative disease- modeling articles. These articles were manually assessed and annotated with disease ontologies, which were then used to classify papers based on disease type. We found that Xenopus is being used to study a diverse array of disease with three main experimental approaches: cell-free egg extracts to study fundamental aspects of cellular and molecular biology, oocytes to study ion transport and channel physiology and embryo experiments focused on congenital diseases. We integrated these data into Xenbase Disease Pages to allow easy navigation to disease information on external databases. Results of this analysis will equip Xenopus researchers with a suite of experimental approaches available to model or dissect a pathological process. Ideally clinicians and basic researchers will use this information to foster collaborations necessary to interrogate the development and treatment of human diseases.
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[This corrects the article DOI: 10.1371/journal.pgen.1002114.].
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Xenbase is the Xenopus model organism database ( www.xenbase.org ), a web-accessible resource that integrates the diverse genomic and biological data for Xenopus research. It hosts a variety of content including current and archived genomes for both X. laevis and X. tropicalis, bioinformatic tools for comparative genetic analyses including BLAST and GBrowse, annotated Xenopus literature, and catalogs of reagents including antibodies, ORFeome clones, morpholinos, and transgenic lines. Xenbase compiles gene-specific pages which include manually curated gene expression images, functional information including gene ontology (GO), disease associations, and links to other major data sources such as NCBI:Entrez, UniProtKB, and Ensembl. We also maintain the Xenopus Anatomy Ontology (XAO) which describes anatomy throughout embryonic development. This chapter provides a full description of the many features of Xenbase, and offers a guide on how to use various tools to perform a variety of common tasks such as identifying nucleic acid or protein sequences, finding gene expression patterns for specific genes, stages or tissues, identifying literature on a specific gene or tissue, locating useful reagents and downloading our extensive content, including Xenopus gene-Human gene disease mapping files.
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Bases de Dados Genéticas , Expressão Gênica , Genoma , Genômica , Xenopus laevis/genética , Animais , Biologia Computacional/métodos , Ontologia Genética , Genômica/métodos , Software , Interface Usuário-Computador , NavegadorRESUMO
Although it is generally accepted that cellular differentiation requires changes to transcriptional networks, dynamic regulation of promoters and enhancers at specific sets of genes has not been previously studied en masse. Exploiting the fact that active promoters and enhancers are transcribed, we simultaneously measured their activity in 19 human and 14 mouse time courses covering a wide range of cell types and biological stimuli. Enhancer RNAs, then messenger RNAs encoding transcription factors, dominated the earliest responses. Binding sites for key lineage transcription factors were simultaneously overrepresented in enhancers and promoters active in each cellular system. Our data support a highly generalizable model in which enhancer transcription is the earliest event in successive waves of transcriptional change during cellular differentiation or activation.
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Diferenciação Celular/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sítios de Ligação , Bovinos , Cães , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Células-Tronco/metabolismoRESUMO
Mp is an irradiation-induced mouse mutation associated with microphthalmia, micropinna and hind limb syndactyly. We show that Mp is caused by a 660 kb balanced inversion on chromosome 18 producing reciprocal 3-prime gene fusion events involving Fbn2 and Isoc1. The Isoc1-Fbn2 fusion gene (Isoc1(Mp)) mRNA has a frameshift and early stop codon resulting in nonsense mediated decay. Homozygous deletions of Isoc1 do not support a significant developmental role for this gene. The Fbn2-Isoc1 fusion gene (Fbn2 (Mp)) predicted protein consists of the N-terminal Fibrillin-2 (amino acids 1-2646, exons 1-62) lacking the C-terminal furin-cleavage site with a short out-of-frame extension encoded by the final exon of Isoc1. The Mp limb phenotype is consistent with that reported in Fbn2 null embryos. However, severe eye malformations, a defining feature of Mp, are not seen in Fbn2 null animals. Fibrillin-2(Mp) forms large fibrillar structures within the rough endoplasmic reticulum (rER) associated with an unfolded protein response and quantitative mass spectrometry shows a generalised defect in protein secretion in conditioned media from mutant cells. In the embryonic eye Fbn2 is expressed within the peripheral ciliary margin (CM). Mp embryos show reduced canonical Wnt-signalling in the CM - known to be essential for ciliary body development - and show subsequent aplasia of CM-derived structures. We propose that the Mp "worse-than-null" eye phenotype plausibly results from a failure in normal trafficking of proteins that are co-expressed with Fbn2 within the CM. The prediction of similar trans-acting protein effects will be an important challenge in the medical interpretation of human mutations from whole exome sequencing.
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Anormalidades do Olho/genética , Proteínas dos Microfilamentos/genética , Microftalmia/genética , Mutação/efeitos da radiação , Animais , Inversão Cromossômica/genética , Cromossomos Humanos Par 18/genética , Éxons , Olho/crescimento & desenvolvimento , Olho/fisiopatologia , Anormalidades do Olho/fisiopatologia , Fibrilina-2 , Fibrilinas , Mutação da Fase de Leitura , Humanos , Camundongos , Microftalmia/fisiopatologia , Fenótipo , Sindactilia/genética , Sindactilia/fisiopatologia , Via de Sinalização Wnt/genéticaRESUMO
PURPOSE OF REVIEW: Uncertainty surrounding medical decision-making is particularly important during end-of-life decision-making. Doubts about the patient's best interests and prognostic accuracy may lead to conflict. RECENT FINDINGS: Many authors have suggested recently that medical attitudes to uncertainty need review. It is inappropriate to avoid discussion of uncertainty during end-of-life care and American literature suggests that patients and families accept uncertainty in end-of-life discussions. Recently, authors have advocated the concept of 'Practical Certainty' accepting that absolute certainty is rarely possible in end-of-life decision-making and openly acknowledging that the physicians are as certain as they can be in the circumstances. Allowing time to provide acceptance of a palliative care pathway and using the collective wisdom of colleagues improves the accuracy of prediction and reduces conflict at the end of life. SUMMARY: The implications of this review are that doctors should not avoid discussing uncertainty in end-of-life conversations and the article provides some recommendations for minimizing conflict arising from end-of-life discussion.
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Atitude do Pessoal de Saúde , Família , Unidades de Terapia Intensiva/ética , Assistência Terminal , Planejamento Antecipado de Cuidados , Comunicação , Conflito de Interesses , Tomada de Decisões , Família/psicologia , Feminino , Humanos , Cuidados para Prolongar a Vida , Masculino , Relações Médico-Paciente , Prognóstico , Assistência Terminal/ética , Assistência Terminal/psicologia , IncertezaRESUMO
LINE-1 (L1) retrotransposons are mobile genetic elements comprising ~17% of the human genome. New L1 insertions can profoundly alter gene function and cause disease, though their significance in cancer remains unclear. Here, we applied enhanced retrotransposon capture sequencing (RC-seq) to 19 hepatocellular carcinoma (HCC) genomes and elucidated two archetypal L1-mediated mechanisms enabling tumorigenesis. In the first example, 4/19 (21.1%) donors presented germline retrotransposition events in the tumor suppressor mutated in colorectal cancers (MCC). MCC expression was ablated in each case, enabling oncogenic ß-catenin/Wnt signaling. In the second example, suppression of tumorigenicity 18 (ST18) was activated by a tumor-specific L1 insertion. Experimental assays confirmed that the L1 interrupted a negative feedback loop by blocking ST18 repression of its enhancer. ST18 was also frequently amplified in HCC nodules from Mdr2(-/-) mice, supporting its assignment as a candidate liver oncogene. These proof-of-principle results substantiate L1-mediated retrotransposition as an important etiological factor in HCC.
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Carcinoma Hepatocelular/genética , Análise Mutacional de DNA , Genes Supressores de Tumor , Neoplasias Hepáticas/genética , Elementos Nucleotídeos Longos e Dispersos , Mutagênese Insercional , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Assessing the appropriateness of continuing life support is a difficult task for intensive care unit staff. Part of this difficulty relates to prognostic uncertainty and the varying reliability of clinical decisions. Uncertainty about prognosis is quickly recognised by patients and families, and can be a source of mistrust and potential conflict. We discuss the reasons for uncertainty and outline key measures to reduce and manage such uncertainty. Practical certainty, where the clinicians are as certain as they can be, with both prognostication and knowledge of patient wishes, may be an appropriate concept for physicians engaged in end-of-life decisions. It involves accurate prognostication, informed surrogates, advance care planning, time to assess response, and the collective wisdom of experienced clinicians. The family conference should develop an agreed plan through shared decision making. The collective wisdom of experienced health care workers with good communication skills and informed patient advocates increases the likelihood of achieving practical certainty and the best decisions. However, greater time and effort seems to be required to improve end-of-life care in the ICU.
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Planejamento Antecipado de Cuidados , Tomada de Decisões , Assistência Terminal , Família , Humanos , Unidades de Terapia Intensiva , Cuidados para Prolongar a Vida , Relações Médico-Paciente , IncertezaRESUMO
Structure-activity relationships (SARs) refer to the relation between chemical structure and pharmacologic activity for a series of compounds. Since the pioneering work of Crum-Brown and Fraser in 1868, they have been increasingly used in the pharmaceutical, chemical and cosmetic industries, especially for drug and chemical design purposes. Structure-activity relationships may be based on various techniques, ranging from considerations of similarity or diversity of molecules to mathematical relationships linking chemical structures to measured activities, the latter being referred to as quantitative SAR or QSAR. This review aims at briefly reviewing the history of SARs and highlighting their interest in delayed and immediate drug allergy using selected examples from the literature. Studies of SAR are commonly conducted in the area of contact dermatitis, a delayed hypersensitivity reaction, to determine the allergenic potential of a given compound without animal testing. In immediate, immunoglobulin E-mediated drug hypersensitivity, this kind of approach remains rather confidential. It has been mainly applied to neuromuscular blocking drugs (muscle relaxants) and betalactam antibiotics (penicillins, cephalosporins). This review shows that SARs can prove useful to (i) predict the allergenic potential of a chemical or a drug, (ii) help identify putative antigenic determinants for each patient or small group of patients sharing the same cross-reactivity pattern, and (iii) predict the likelihood of adverse reactions to related molecules and select safe alternatives.
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Desenho de Fármacos , Hipersensibilidade a Drogas/etiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Animais , Reações Cruzadas/imunologia , Dermatite de Contato/etiologia , Dermatite de Contato/imunologia , Hipersensibilidade a Drogas/imunologia , Humanos , Imunoglobulina E/imunologia , Preparações Farmacêuticas/química , Relação Quantitativa Estrutura-Atividade , Relação Estrutura-Atividade , Fatores de TempoRESUMO
Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1(tm1a)) that reduces mRNA to â¼10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1(tm1a/tm1a)). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1(tm1a/tm1a) embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice.
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Anoftalmia/genética , Proteína Morfogenética Óssea 1/antagonistas & inibidores , Mutação , Osteonectina , Síndrome de Waardenburg/genética , Animais , Proteína Morfogenética Óssea 1/genética , Coloboma/genética , Análise Mutacional de DNA , Extremidades/crescimento & desenvolvimento , Olho/crescimento & desenvolvimento , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Osteonectina/genética , Osteonectina/metabolismo , Linhagem , Sindactilia/genética , Xenopus laevisRESUMO
Regulation of cell proliferation and motility is essential for normal development. The Rho family of GTPases plays a critical role in the control of cell polarity and migration by effecting the cytoskeleton, membrane trafficking, and cell adhesion. We investigated a recognized developmental disorder, Adams-Oliver syndrome (AOS), characterized by the combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). Through a genome-wide linkage analysis, we detected a locus for autosomal-dominant ACC-TTLD on 3q generating a maximum LOD score of 4.93 at marker rs1464311. Candidate-gene- and exome-based sequencing led to the identification of independent premature truncating mutations in the terminal exon of the Rho GTPase-activating protein 31 gene, ARHGAP31, which encodes a Cdc42/Rac1 regulatory protein. Mutant transcripts are stable and increase ARHGAP31 activity in vitro through a gain-of-function mechanism. Constitutively active ARHGAP31 mutations result in a loss of available active Cdc42 and consequently disrupt actin cytoskeletal structures. Arhgap31 expression in the mouse is substantially restricted to the terminal limb buds and craniofacial processes during early development; these locations closely mirror the sites of impaired organogenesis that characterize this syndrome. These data identify the requirement for regulated Cdc42 and/or Rac1 signaling processes during early human development.
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Displasia Ectodérmica/genética , Proteínas Ativadoras de GTPase/genética , Mutação , Actinas/metabolismo , Adesão Celular , Movimento Celular , Polaridade Celular , Proliferação de Células , Mapeamento Cromossômico , Citoesqueleto/metabolismo , Análise Mutacional de DNA , Displasia Ectodérmica/embriologia , Feminino , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Deformidades Congênitas dos Membros/embriologia , Deformidades Congênitas dos Membros/genética , Masculino , Dermatoses do Couro Cabeludo/congênito , Dermatoses do Couro Cabeludo/embriologia , Dermatoses do Couro Cabeludo/genética , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Sonic hedgehog (Shh) signalling controls integrated specification of digit pattern and growth in the chick wing but downstream gene networks remain to be unravelled. We analysed 3D expression patterns of genes encoding cell cycle regulators using Optical Projection Tomography. Hierarchical clustering of spatial matrices of gene expression revealed a dorsal layer of the wing bud, in which almost all genes were expressed, and that genes encoding positive cell cycle regulators had similar expression patterns while those of N-myc and CyclinD2 were distinct but closely related. We compared these patterns computationally with those of genes implicated in digit specification and Ptch1, 50 genes in total. Nineteen genes have similar posterior expression to Ptch1, including Hoxd13, Sall1, Hoxd11, and Bmp2, all likely Gli targets in mouse limb, and cell cycle genes, N-myc, CyclinD2. We suggest that these genes contribute to a network integrating digit specification and growth in response to Shh.
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Extremidades/embriologia , Genes cdc/fisiologia , Asas de Animais/embriologia , Asas de Animais/metabolismo , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Embrião de Galinha , Galinhas , Extremidades/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Hoxd13, Tbx2, Tbx3, Sall1 and Sall3 genes are candidates for encoding antero-posterior positional values in the developing chick wing and specifying digit identity. In order to build up a detailed profile of gene expression patterns in cell lineages that give rise to each of the digits over time, we compared 3 dimensional (3D) expression patterns of these genes during wing development and related them to digit fate maps. 3D gene expression data at stages 21, 24 and 27 spanning early bud to digital plate formation, captured from in situ hybridisation whole mounts using Optical Projection Tomography (OPT) were mapped to reference wing bud models. Grafts of wing bud tissue from GFP chicken embryos were used to fate map regions of the wing bud giving rise to each digit; 3D images of the grafts were captured using OPT and mapped on to the same models. Computational analysis of the combined computerised data revealed that Tbx2 and Tbx3 are expressed in digit 3 and 4 progenitors at all stages, consistent with encoding stable antero-posterior positional values established in the early bud; Hoxd13 and Sall1 expression is more dynamic, being associated with posterior digit 3 and 4 progenitors in the early bud but later becoming associated with anterior digit 2 progenitors in the digital plate. Sox9 expression in digit condensations lies within domains of digit progenitors defined by fate mapping; digit 3 condensations express Hoxd13 and Sall1, digit 4 condensations Hoxd13, Tbx3 and to a lesser extent Tbx2. Sall3 is only transiently expressed in digit 3 progenitors at stage 24 together with Sall1 and Hoxd13; then becomes excluded from the digital plate. These dynamic patterns of expression suggest that these genes may play different roles in digit identity either together or in combination at different stages including the digit condensation stage.
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Padronização Corporal/genética , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Imageamento Tridimensional , Fatores de Transcrição/genética , Asas de Animais/embriologia , Asas de Animais/metabolismo , Animais , Desenvolvimento Ósseo/genética , Linhagem da Célula/genética , Embrião de Galinha , Biologia Computacional , Proteínas de Fluorescência Verde/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Congenital anomalies of the kidney and urinary tract (CAKUTs) are common disorders of human development affecting the renal parechyma, renal pelvis, ureter, bladder and urethra; they show evidence of shared genetic aetiology, although the molecular basis of this remains unknown in the majority of cases. Breakpoint mapping of a de novo, apparently balanced, reciprocal translocation associated with bilateral renal agenesis has implicated the gene encoding the nuclear steroid hormone receptor ESRRG as a candidate gene for CAKUT. Here we show that the Esrrg protein is detected throughout early ureteric ducts as cytoplasmic/sub-membranous staining; with nuclear localization seen in developing nephrons. In 14.5-16.5 dpc (days post-conception) mouse embryos, Esrrg localizes to the subset of ductal tissue within the kidney, liver and lung. The renal ductal expression becomes localized to renal papilla by 18.5 dpc. Perturbation of function was performed in embryonic mouse kidney culture using pooled siRNA to induce knock-down and a specific small-molecule agonist to induce aberrant activation of Esrrg. Both resulted in severe abnormality of early branching events of the ureteric duct. Mouse embryos with a targeted inactivation of Esrrg on both alleles (Esrrg(-/-)) showed agenesis of the renal papilla but normal development of the cortex and remaining medulla. Taken together, these results suggest that Esrrg is required for early branching events of the ureteric duct that occur prior to the onset of nephrogenesis. These findings confirm ESRRG as a strong candidate gene for CAKUT.
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Medula Renal/embriologia , Receptores de Estrogênio/metabolismo , Ureter/embriologia , Ureter/metabolismo , Animais , Anormalidades Congênitas/embriologia , Anormalidades Congênitas/genética , Anormalidades Congênitas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Rim/anormalidades , Rim/embriologia , Rim/metabolismo , Nefropatias/congênito , Medula Renal/metabolismo , Camundongos , Camundongos Knockout , Organogênese , Receptores de Estrogênio/genéticaRESUMO
Sonic hedgehog (Shh) signalling by the polarizing region at the posterior margin of the chick wing bud is pivotal in patterning the digits but apart from a few key downstream genes, such as Hoxd13, which is expressed in the posterior region of the wing that gives rise to the digits, the genes that mediate the response to Shh signalling are not known. To find genes that are co-expressed with Hoxd13 in the posterior of chick wing buds and regulated in the same way, we used microarrays to compare gene expression between anterior and posterior thirds of wing buds from normal chick embryos and from polydactylous talpid³ mutant chick embryos, which have defective Shh signalling due to lack of primary cilia. We identified 1070 differentially expressed gene transcripts, which were then clustered. Two clusters contained genes predominantly expressed in posterior thirds of normal wing buds; in one cluster, genes including Hoxd13, were expressed at high levels in anterior and posterior thirds in talpid³ wing buds, in the other cluster, genes including Ptc1, were expressed at low levels in anterior and posterior thirds in talpid³ wing buds. Expression patterns of genes in these two clusters were validated in normal and talpid³ mutant wing buds by in situ hybridisation and demonstrated to be responsive to application of Shh. Expression of several genes in the Hoxd13 cluster was also shown to be responsive to manipulation of protein kinase A (PKA) activity, thus demonstrating regulation by Gli repression. Genes in the Hoxd13 cluster were then sub-clustered by computational comparison of 3D expression patterns in normal wing buds to produce syn-expression groups. Hoxd13 and Sall1 are syn-expressed in the posterior region of early chick wing buds together with 6 novel genes which are likely to be functionally related and represent secondary targets of Shh signalling. Other groups of syn-expressed genes were also identified, including a group of genes involved in vascularisation.
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Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/metabolismo , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Transdução de Sinais/genética , Asas de Animais/embriologia , Asas de Animais/metabolismo , Animais , Embrião de Galinha , Análise por Conglomerados , Redes Reguladoras de Genes/genética , Proteínas de Homeodomínio/genética , Família Multigênica/genética , Receptores Patched , Receptores de Superfície Celular/genética , Proteínas Repressoras/metabolismo , Reprodutibilidade dos TestesRESUMO
EMAGE (http://www.emouseatlas.org/emage) is a freely available online database of in situ gene expression patterns in the developing mouse embryo. Gene expression domains from raw images are extracted and integrated spatially into a set of standard 3D virtual mouse embryos at different stages of development, which allows data interrogation by spatial methods. An anatomy ontology is also used to describe sites of expression, which allows data to be queried using text-based methods. Here, we describe recent enhancements to EMAGE including: the release of a completely re-designed website, which offers integration of many different search functions in HTML web pages, improved user feedback and the ability to find similar expression patterns at the click of a button; back-end refactoring from an object oriented to relational architecture, allowing associated SQL access; and the provision of further access by standard formatted URLs and a Java API. We have also increased data coverage by sourcing from a greater selection of journals and developed automated methods for spatial data annotation that are being applied to spatially incorporate the genome-wide (approximately 19,000 gene) 'EURExpress' dataset into EMAGE.
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Biologia Computacional/métodos , Bases de Dados Genéticas , Bases de Dados de Ácidos Nucleicos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Expressão Gênica , Acesso à Informação , Animais , Automação , Biologia Computacional/tendências , Desenvolvimento Embrionário/genética , Armazenamento e Recuperação da Informação/métodos , Internet , Camundongos , Linguagens de Programação , SoftwareRESUMO
BACKGROUND: The authors observed a patient with a cryptic subtelomeric de novo balanced translocation 46,XY.ish t(11;20)(p15.4;q13.2) presenting with severe mental retardation, muscular hypotonia, seizures, bilateral sensorineural hearing loss, submucous cleft palate, persistent ductus Botalli, unilateral cystic kidney dysplasia and frequent infections. METHODS AND RESULTS: Fluorescence in situ hybridisation mapping and sequencing of the translocation breakpoints showed that no known genes are disrupted at 20q13.2 and that ST5 (suppression of tumorigenicity 5; MIM 140750) is disrupted on 11p15.4. By quantitative PCR from different human tissues, the authors found ST5 to be relatively evenly expressed in fetal tissues. ST5 expression was more pronounced in adult brain, kidney and muscle than in the corresponding fetal tissues, whereas expression in other tissues was generally lower than in the fetal tissue. Using RNA in situ hybridisation in mouse, the authors found that St5 is expressed in the frontal cortex during embryonic development. In adult mouse brain, expression of St5 was especially high in the hippocampal area and cerebellum. CONCLUSION: Hence, the authors suppose that ST5 plays an important role in central nervous system development probably due to disturbance of DENN-domain-mediated vesicle formation and neurotransmitter trafficking. Thus, these findings implicate ST5 in the aetiology of mental retardation, seizures and multiple congenital anomalies.
Assuntos
Anormalidades Múltiplas/genética , Proteínas de Ligação a DNA/genética , Deficiência Intelectual/genética , Proteínas Supressoras de Tumor/genética , Animais , Pré-Escolar , Pontos de Quebra do Cromossomo , Mapeamento Cromossômico , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Dosagem de Genes , Histocitoquímica , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Especificidade de Órgãos , RNA , Tomografia Óptica , Proteínas Supressoras de Tumor/metabolismoRESUMO
Pierre Robin sequence (PRS) is an important subgroup of cleft palate. We report several lines of evidence for the existence of a 17q24 locus underlying PRS, including linkage analysis results, a clustering of translocation breakpoints 1.06-1.23 Mb upstream of SOX9, and microdeletions both approximately 1.5 Mb centromeric and approximately 1.5 Mb telomeric of SOX9. We have also identified a heterozygous point mutation in an evolutionarily conserved region of DNA with in vitro and in vivo features of a developmental enhancer. This enhancer is centromeric to the breakpoint cluster and maps within one of the microdeletion regions. The mutation abrogates the in vitro enhancer function and alters binding of the transcription factor MSX1 as compared to the wild-type sequence. In the developing mouse mandible, the 3-Mb region bounded by the microdeletions shows a regionally specific chromatin decompaction in cells expressing Sox9. Some cases of PRS may thus result from developmental misexpression of SOX9 due to disruption of very-long-range cis-regulatory elements.