Integrated analysis of Wnt signalling system component gene expression.

Wnt signalling controls patterning and differentiation across many tissues and organs of the developing embryo through temporally and spatially restricted expression of multi-gene families encoding ligands, receptors, pathway modulators and intracellular components. Here, we report an integrated analysis of key genes in the 3D space of the mouse embryo across multiple stages of development. We applied a method for 3D/3D image transformation to map all gene expression patterns to a single reference embryo for each stage, providing both visual analysis and volumetric mapping allowing computational methods to interrogate the combined expression patterns. We identify territories where multiple Wnt and Fzd genes are co-expressed and cross-compare all patterns, including all seven Wnt paralogous gene pairs. The comprehensive analysis revealed regions in the embryo where no Wnt or Fzd gene expression is detected, and where single Wnt genes are uniquely expressed. This work provides insight into a previously unappreciated level of organisation of expression patterns, as well as presenting a resource that can be utilised further by the research community for whole-system analysis.

Nerves of Steel: a Low-Cost Method for 3D Printing the Cranial Nerves.

Steady-state free precession (SSFP) magnetic resonance imaging (MRI) can demonstrate details down to the cranial nerve (CN) level. High-resolution three-dimensional (3D) visualization can now quickly be performed at the workstation. However, we are still limited by visualization on flat screens. The emerging technologies in rapid prototyping or 3D printing overcome this limitation. It comprises a variety of automated manufacturing techniques, which use virtual 3D data sets to fabricate solid forms in a layer-by-layer technique. The complex neuroanatomy of the CNs may be better understood and depicted by the use of highly customizable advanced 3D printed models. In this technical note, after manually perfecting the segmentation of each CN and brain stem on each SSFP-MRI image, initial 3D reconstruction was performed. The bony skull base was also reconstructed from computed tomography (CT) data. Autodesk 3D Studio Max, available through freeware student/educator license, was used to three-dimensionally trace the 3D reconstructed CNs in order to create smooth graphically designed CNs and to assure proper fitting of the CNs into their respective neural foramina and fissures. This model was then 3D printed with polyamide through a commercial online service. Two different methods are discussed for the key segmentation and 3D reconstruction steps, by either using professional commercial software, i.e., Materialise Mimics, or utilizing a combination of the widely available software Adobe Photoshop, as well as a freeware software, OsiriX Lite.

Representing kidney development using the gene ontology.

Gene Ontology (GO) provides dynamic controlled vocabularies to aid in the description of the functional biological attributes and subcellular locations of gene products from all taxonomic groups (www.geneontology.org). Here we describe collaboration between the renal biomedical research community and the GO Consortium to improve the quality and quantity of GO terms describing renal development. In the associated annotation activity, the new and revised terms were associated with gene products involved in renal development and function. This project resulted in a total of 522 GO terms being added to the ontology and the creation of approximately 9,600 kidney-related GO term associations to 940 UniProt Knowledgebase (UniProtKB) entries, covering 66 taxonomic groups. We demonstrate the impact of these improvements on the interpretation of GO term analyses performed on genes differentially expressed in kidney glomeruli affected by diabetic nephropathy. In summary, we have produced a resource that can be utilized in the interpretation of data from small- and large-scale experiments investigating molecular mechanisms of kidney function and development and thereby help towards alleviating renal disease.

EMAGE: Electronic Mouse Atlas of Gene Expression.

The EMAGE (Electronic Mouse Atlas of Gene Expression) database (http://www.emouseatlas.org/emage) allows users to perform on-line queries of mouse developmental gene expression. EMAGE data are represented spatially using a framework of 3D mouse embryo models, thus allowing uniquely spatial queries to be carried out alongside more traditional text-based queries. This spatial representation of the data also allows a comparison of spatial similarity between the expression patterns. The data are mapped to the models by a team of curators using bespoke mapping software, and the associated meta-data are curated for accuracy and completeness. The data contained in EMAGE are gathered from three main sources: from the published literature, through large-scale screens and collaborations, and via direct submissions from researchers. There are a variety of ways to query the EMAGE database via the on-line search interfaces, as well as via direct computational script-based queries. EMAGE is a free, on-line, community resource funded by the Medical Research Council, UK.

EMAP/EMAPA ontology of mouse developmental anatomy: 2013 update.

BACKGROUND: The Edinburgh Mouse Atlas Project (EMAP) ontology of mouse developmental anatomy provides a standard nomenclature for describing normal and mutant mouse embryo anatomy. The ontology forms the core of the EMAP atlas and is used for annotating gene expression data by the mouse Gene Expression Database (GXD), Edinburgh Mouse Atlas of Gene Expression (EMAGE) and other database resources. FINDINGS: The original EMAP ontology listed anatomical entities for each developmental stage separately, presented as uniparental graphs organized as a strict partonomy. An "abstract" (i.e. non-stage-specific) representation of mouse developmental anatomy has since been developed. In this version (EMAPA) all instances for a given anatomical entity are presented as a single term, together with the first and last stage at which it is considered to be present. Timed-component anatomies are now derived using staging information in the "primary" non-timed version. Anatomical entities are presented as a directed acyclic graph enabling multiple parental relationships. Subsumption classification as well as partonomic and other types of relationships can now be represented. Most concept names are unique, with compound names constructed using standardized nomenclature conventions, and alternative names associated as synonyms. CONCLUSIONS: The ontology has been extended and refined in a collaborative effort between EMAP and GXD, with additional input from others. Efforts are also underway to improve the revision process with regards to updating and editorial control. The revised EMAPA ontology is freely available from the OBO Foundry resource, with descriptive information and other documentation presented in associated Wiki pages (http://www.obofoundry.org/wiki/index.php/EMAPA:Main_Page).

Genetic ancestry and the relationship of cigarette smoking to lung function and per cent emphysema in four race/ethnic groups: a cross-sectional study.

BACKGROUND: Cigarette smoking is the major cause of chronic obstructive pulmonary disease and emphysema. Recent studies suggest that susceptibility to cigarette smoke may vary by race/ethnicity; however, they were generally small and relied on self-reported race/ethnicity. OBJECTIVE: To test the hypothesis that relationships of smoking to lung function and per cent emphysema differ by genetic ancestry and self-reported race/ethnicity among Caucasians, African-Americans, Hispanics and Chinese-Americans. DESIGN: Cross-sectional population-based study of adults age 45-84 years in the USA. MEASUREMENTS: Principal components of genetic ancestry and continental ancestry estimated from one million genome-wide single nucleotide polymorphisms; pack-years of smoking; spirometry measured for 3344 participants; and per cent emphysema on computed tomography for 8224 participants. RESULTS: The prevalence of ever-smoking was: Caucasians, 57.6%; African-Americans, 56.4%; Hispanics, 46.7%; and Chinese-Americans, 26.8%. Every 10 pack-years was associated with -0.73% (95% CI -0.90% to -0.56%) decrement in the forced expiratory volume in 1 s to forced vital capacity (FEV1 to FVC) and a 0.23% (95% CI 0.08% to 0.38%) increase in per cent emphysema. There was no evidence that relationships of pack-years to the FEV1 to FVC, airflow obstruction and per cent emphysema varied by genetic ancestry (all p>0.10), self-reported race/ethnicity (all p>0.10) or, among African-Americans, African ancestry. There were small differences in relationships of pack-years to the FEV1 among male Chinese-Americans and to the FEV1 to FVC ratio with African and Native American ancestry among male Hispanics only. CONCLUSIONS: In this large cohort, there was little to no evidence that the associations of smoking to lung function and per cent emphysema differed by genetic ancestry or self-reported race/ethnicity.

eChickAtlas: an introduction to the database.

The precise control of gene expression is critical in embryonic development. Quantitative assays, such as microarrays and RNA sequencing, provide gene expression levels for a large number of genes, but do not contain spatial information. In contrast, in situ methods, such as in situ hybridization and immunohistochemistry, provide spatial resolution, but poor quantification and can only reveal the expression of one, or very few genes at a time. Furthermore, the usual methods of documenting the results, by photographing whole mounts or sections, makes it very difficult to assess the three-dimensional (3D) relationships between expressing and nonexpressing cells. Optical projection tomography (OPT) can capture the full 3D expression pattern in a whole embryo at a reasonable level of resolution and at moderately high throughput. A large database containing spatio-temporal patterns of expression for the mouse (e-Mouse Atlas Project, EMAP, www.emouseatlas.org) has been created, incorporating 3D information. Like the mouse, the chick is an important model in developmental biology and translational studies. To facilitate comparisons between these important model organisms, we have created a 3D anatomical atlas, accompanied by an anatomical ontology of the chick embryo and a database of gene expression patterns during chick development. This database is publicly available (www.echickatlas.org).

Differential deployment of paralogous Wnt genes in the mouse and chick embryo during development.

Genes encoding Wnt ligands are crucial in body patterning and are highly conserved among metazoans. Given their conservation at the protein-coding level, it is likely that changes in where and when these genes are active are important in generating evolutionary variations. However, we lack detailed knowledge about how their deployment has diverged. Here, we focus on four Wnt subfamilies (Wnt2, Wnt5, Wnt7, and Wnt8) in mammalian and avian species, consisting of a paralogous gene pair in each, believed to have duplicated in the last common ancestor of vertebrates. We use three-dimensional imaging to capture expression patterns in detail and carry out systematic comparisons. We find evidence of greater divergence between these subgroup paralogues than the respective orthologues, consistent with some level of subfunctionalization/neofunctionalization in the common vertebrate ancestor that has been conserved. However, there were exceptions; in the case of chick Wnt2b, individual sites were shared with both mouse Wnt2 and Wnt2b. We also find greater divergence, between paralogues and orthologues, in some subfamilies (Wnt2 and Wnt8) compared to others (Wnt5 and Wnt7) with the more highly similar expression patterns showing more extensive expression in more structures in the embryo. Wnt8 genes were most restricted and most divergent. Major sites of expression for all subfamilies include CNS, limbs, and facial region, and in general there were more similarities in gene deployment in these territories with divergent patterns featuring more in organs such as heart and gut. A detailed comparison of gene expression patterns in the limb showed similarities in overall combined domains across species with notable differences that may relate to lineage-specific morphogenesis.

eMouseAtlas, EMAGE, and the spatial dimension of the transcriptome.

eMouseAtlas (www.emouseatlas.org) is a comprehensive online resource to visualise mouse development and investigate gene expression in the mouse embryo. We have recently deployed a completely redesigned Mouse Anatomy Atlas website (www.emouseatlas.org/emap/ema) that allows users to view 3D embryo reconstructions, delineated anatomy, and high-resolution histological sections. A new feature of the website is the IIP3D web tool that allows a user to view arbitrary sections of 3D embryo reconstructions using a web browser. This feature provides interactive access to very high-volume 3D images via a tiled pan-and-zoom style interface and circumvents the need to download large image files for visualisation. eMouseAtlas additionally includes EMAGE (Edinburgh Mouse Atlas of Gene Expression) (www.emouseatlas.org/emage), a freely available, curated online database of in situ gene expression patterns, where gene expression domains extracted from raw data images are spatially mapped into atlas embryo models. In this way, EMAGE introduces a spatial dimension to transcriptome data and allows exploration of the spatial similarity between gene expression patterns. New features of the EMAGE interface allow complex queries to be built, and users can view and compare multiple gene expression patterns. EMAGE now includes mapping of 3D gene expression domains captured using the imaging technique optical projection tomography. 3D mapping uses WlzWarp, an open-source software tool developed by eMouseAtlas.

Access and use of the GUDMAP database of genitourinary development.

The Genitourinary Development Molecular Atlas Project (GUDMAP) aims to document gene expression across time and space in the developing urogenital system of the mouse, and to provide access to a variety of relevant practical and educational resources. Data come from microarray gene expression profiling (from laser-dissected and FACS-sorted samples) and in situ hybridization at both low (whole-mount) and high (section) resolutions. Data are annotated to a published, high-resolution anatomical ontology and can be accessed using a variety of search interfaces. Here, we explain how to run typical queries on the database, by gene or anatomical location, how to view data, how to perform complex queries, and how to submit data.

The GUDMAP database--an online resource for genitourinary research.

The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is an international consortium working to generate gene expression data and transgenic mice. GUDMAP includes data from large-scale in situ hybridisation screens (wholemount and section) and microarray gene expression data of microdissected, laser-captured and FACS-sorted components of the developing mouse genitourinary (GU) system. These expression data are annotated using a high-resolution anatomy ontology specific to the developing murine GU system. GUDMAP data are freely accessible at www.gudmap.org via easy-to-use interfaces. This curated, high-resolution dataset serves as a powerful resource for biologists, clinicians and bioinformaticians interested in the developing urogenital system. This paper gives examples of how the data have been used to address problems in developmental biology and provides a primer for those wishing to use the database in their own research.

EMAGE mouse embryo spatial gene expression database: 2010 update.

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.

Genetic background influences embryonic lethality and the occurrence of neural tube defects in Men1 null mice: relevance to genetic modifiers.

Germline mutations of the multiple endocrine neoplasia type 1 (MEN1) gene cause parathyroid, pancreatic and pituitary tumours in man. MEN1 mutations also cause familial isolated primary hyperparathyroidism (FIHP) and the same MEN1 mutations, in different families, can cause either FIHP or MEN1. This suggests a role for genetic background and modifier genes in altering the expression of a mutation. We investigated the effects of genetic background on the phenotype of embryonic lethality that occurs in a mouse model for MEN1. Men1(+/-) mice were backcrossed to generate C57BL/6 and 129S6/SvEv incipient congenic strains, and used to obtain homozygous Men1(-/-) mice. No viable Men1(-/-) mice were obtained. The analysis of 411 live embryos obtained at 9.5-16.5 days post-coitum (dpc) revealed that significant deviations from the expected Mendelian 1:2:1 genotype ratio were first observed at 12.5 and 14.5 dpc in the 129S6/SvEv and C57BL/6 strains respectively (P<0.05). Moreover, live Men1(-/-) embryos were absent by 13.5 and 15.5 dpc in the 129S6/SvEv and C57BL/6 strains respectively thereby indicating an earlier lethality by 2 days in the 129S6/SvEv strain (P<0.01). Men1(-/-) embryos had macroscopic haemorrhages, and histology and optical projection tomography revealed them to have internal haemorrhages, myocardial hypotrophy, pericardial effusion, hepatic abnormalities and neural tube defects. The neural tube defects occurred exclusively in 129S6/SvEv embryos (21 vs 0%, P<0.01). Thus, our findings demonstrate the importance of genetic background in influencing the phenotypes of embryonic lethality and neural tube defects in Men1(-/-) mice, and implicate a role for genetic modifiers.

Gene expression analysis of canonical Wnt pathway transcriptional regulators during early morphogenesis of the facial region in the mouse embryo.

Structures and features of the face, throat and neck are formed from a series of branchial arches that grow out along the ventrolateral aspect of the embryonic head. Multiple signalling pathways have been implicated in patterning interactions that lead to species-specific growth and differentiation within the branchial region that sculpt these features. A direct role for Wnt signalling in particular has been shown. The spatial and temporal distribution of Wnt pathway components contributes to the operation of the signalling system. We present the precise distribution of gene expression of canonical Wnt pathway transcriptional regulators, Tcf1, Lef1, Tcf3, Tcf4 and beta-catenin between embryonic day (E) 9.5 and 11.5. In situ hybridization combined with Optical Projection Tomography was used to record and compare distribution of transcripts in 3D within the developing branchial arches. This shows widespread yet very specific expression of the gene set indicating that all genes contribute to proper patterning of the region. Tcf1 and Lef1 are more prominent in rostral arches, particularly at later ages, and Tcf3 and Tcf4 are in general expressed more deeply (medial/endodermal aspect) in the arches than Tcf1 and Lef1. Comparison with Wnt canonical pathway readout patterns shows that the relationship between the expression of individual transcription factors and activation of the pathway is not simple, indicating complexity and flexibility in the signalling system.

The Mouse Limb Anatomy Atlas: an interactive 3D tool for studying embryonic limb patterning.

BACKGROUND: The developing mouse limb is widely used as a model system for studying tissue patterning. Despite this, few references are available that can be used for the correct identification of developing limb structures, such as muscles and tendons. Existing textual references consist of two-dimensional (2D) illustrations of the adult rat or mouse limb that can be difficult to apply when attempting to describe the complex three-dimensional (3D) relationship between tissues. RESULTS: To improve the resources available in the mouse model, we have generated a free, web-based, interactive reference of limb muscle, tendon, and skeletal structures at embryonic day (E) 14.5 http://www.nimr.mrc.ac.uk/3dlimb/. The Atlas was generated using mouse forelimb and hindlimb specimens stained using immunohistochemistry to detect muscle and tendon. Limbs were scanned using Optical Projection Tomography (OPT), reconstructed to make 3D models and annotated using computer-assisted segmentation tools in Amira 3D Visualisation software. The annotated dataset is visualised using Java, JAtlasView software. Users click on the names of structures and view their shape, position and relationship with other structures within the 3D model and also in 2D virtual sections. CONCLUSION: The Mouse Limb Anatomy Atlas provides a novel and valuable tool for researchers studying limb development and can be applied to a range of research areas, including the identification of abnormal limb patterning in transgenic lines and studies of models of congenital limb abnormalities. By using the Atlas for "virtual" dissection, this resource offers an alternative to animal dissection. The techniques we have developed and employed are also applicable to many other model systems and anatomical structures.

Organization of the pronephric kidney revealed by large-scale gene expression mapping.

BACKGROUND: The pronephros, the simplest form of a vertebrate excretory organ, has recently become an important model of vertebrate kidney organogenesis. Here, we elucidated the nephron organization of the Xenopus pronephros and determined the similarities in segmentation with the metanephros, the adult kidney of mammals. RESULTS: We performed large-scale gene expression mapping of terminal differentiation markers to identify gene expression patterns that define distinct domains of the pronephric kidney. We analyzed the expression of over 240 genes, which included members of the solute carrier, claudin, and aquaporin gene families, as well as selected ion channels. The obtained expression patterns were deposited in the searchable European Renal Genome Project Xenopus Gene Expression Database. We found that 112 genes exhibited highly regionalized expression patterns that were adequate to define the segmental organization of the pronephric nephron. Eight functionally distinct domains were discovered that shared significant analogies in gene expression with the mammalian metanephric nephron. We therefore propose a new nomenclature, which is in line with the mammalian one. The Xenopus pronephric nephron is composed of four basic domains: proximal tubule, intermediate tubule, distal tubule, and connecting tubule. Each tubule may be further subdivided into distinct segments. Finally, we also provide compelling evidence that the expression of key genes underlying inherited renal diseases in humans has been evolutionarily conserved down to the level of the pronephric kidney. CONCLUSION: The present study validates the Xenopus pronephros as a genuine model that may be used to elucidate the molecular basis of nephron segmentation and human renal disease.

Integrating technologies for comparing 3D gene expression domains in the developing chick limb.

Chick embryos are good models for vertebrate development due to their accessibility and manipulability. Recent large increases in available genomic data from both whole genome sequencing and EST projects provide opportunities for identifying many new developmentally important chicken genes. Traditional methods of documenting when and where specific genes are expressed in embryos using whole amount and section in-situ hybridisation do not readily allow appreciation of 3-dimensional (3D) patterns of expression, but this can be accomplished by the recently developed microscopy technique, Optical Projection Tomography (OPT). Here we show that OPT data on the developing chick wing from different labs can be reliably integrated into a common database, that OPT is efficient in capturing 3D gene expression domains and that such domains can be meaningfully compared. Novel protocols are used to compare 3D expression domains of 7 genes known to be involved in chick wing development. This reveals previously unappreciated relationships and demonstrates the potential, using modern genomic resources, for building a large scale 3D atlas of gene expression. Such an atlas could be extended to include other types of data, such as fate maps, and the approach is also more generally applicable to embryos, organs and tissues.

3D representation of Wnt and Frizzled gene expression patterns in the mouse embryo at embryonic day 11.5 (Ts19).

Wnt signalling is one of the fundamental cell communication systems operating in the embryo and the collection of 19 Wnt and 10 Frizzled (Fzd) receptor genes (in mouse and human) represent just part of a complex system to be unravelled. Here we present a spatially comprehensive set of data on the 3D distribution of Wnt and Fzd gene expression patterns at a carefully selected single stage of mouse development. Overviews and selected features of the patterns are presented and the full 3D data set, generated by fully described probes, is available to the research community through the Edinburgh Mouse Atlas of Gene Expression. In addition to being comprehensive, the data set has been generated and recorded in a consistent manner to facilitate comparisons between gene expression patterns with the capacity to generate matching virtual sections from the 3D representations for specific studies. Expression patterns in the left forelimb were selected for more detailed comparative description. In addition to confirming the previously published expression of these genes, our whole embryo and limb bud analyses significantly extend the data in terms of details of the patterns and the addition of previously undetected sites of expression. Our focussed analysis of expression domains in the limb, defined by just two gene families, reveals a surprisingly high degree of spatial complexity and underlines the enormous potential for local cellular interactions that exist within an emerging structure. This work also highlights the use of OPT to generate detailed high-quality, spatially complex expression data that is readily comparable between specimens and can be reviewed and reanalysed as required for specific studies. It represents a core set of data that will be extended with additional stages of development and through addition of potentially interacting genes and ultimately other cross-regulatory communication pathways operating in the embryo.

Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE).

One purpose of the biomedical literature is to report results in sufficient detail that the methods of data collection and analysis can be independently replicated and verified. Here we present reporting guidelines for gene expression localization experiments: the minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). MISFISHIE is modeled after the Minimum Information About a Microarray Experiment (MIAME) specification for microarray experiments. Both guidelines define what information should be reported without dictating a format for encoding that information. MISFISHIE describes six types of information to be provided for each experiment: experimental design, biomaterials and treatments, reporters, staining, imaging data and image characterizations. This specification has benefited the consortium within which it was developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal.