A Digital Framework to Build,Visualize and Analyze a Gene Expression Atlas with Cellular Resolution in Zebrafish Early Embryogenesis

A gene expression atlas is an essential resource to quantify and understand the multiscale processes of embryogenesis in time and space. The automated reconstruction of a prototypic 4D atlas for vertebrate early embryos, using multicolor fluorescence in situ hybridization with nuclear counterstain, requires dedicated computational strategies. To this goal, we designed an original methodological framework implemented in a software tool called Match-IT. With only minimal human supervision, our system is able to gather gene expression patterns observed in different analyzed embryos with phenotypic variability and map them onto a series of common 3D templates over time, creating a 4D atlas. This framework was used to construct an atlas composed of 6 gene expression templates from a cohort of zebrafish early embryos spanning 6 developmental stages from 4 to 6.3 hpf (hours post fertilization). They included 53 specimens, 181,415 detected cell nuclei and the segmentation of 98 gene expression patterns observed in 3D for 9 different genes. In addition, an interactive visualization software, Atlas-IT, was developed to inspect, supervise and analyze the atlas. Match-IT and Atlas-IT, including user manuals, representative datasets and video tutorials, are publicly and freely available online. We also propose computational methods and tools for the quantitative assessment of the gene expression templates at the cellular scale, with the identification, visualization and analysis of coexpression patterns, synexpression groups and their dynamics through developmental stages.     

Methods for acquisition of quantitative from confocal images of gene expression in situ

In this review we summarize original methods for the extraction quantitative information from the confocal images of gene expression patterns. These methods include image segmentation, extraction of quantitative numerical data on gene expression, removal of background signal and spatial registration. Finally it is possible to construct a spatiotemporal atlas of gene expression form individual images obtained at each developmental stage. Initially all methods were developed to extract quantitative numerical information form confocal images of segmentation gene expression in Drosophila melanogaster. Application of these methods to Drosophila images makes it possible to reveal new mechanisms of formation of segmentation gene expression domains, as well as to construct the quantitative atlas of segmentation gene expression. Most image processing procedures can be easily adapted to process a wide range of biological images.     

The European Renal Genome Project

Rapid progress in genome research creates a wealth of information on the functional annotation of mammalian genome sequences. However, as we accumulate large amounts of scientific information we are facing problems of how to integrate and relate the data produced by various genomic approaches. Here, we propose a novel concept of an organ atlas where diverse data from expression maps to histological findings to mutant phenotypes can be queried, compared and visualized in the context of a three dimensional reconstruction of the organ. We will seek proof of concept for the organ atlas by elucidating genetic pathways involved in development and pathophysiology of the kidney. Such a kidney atlas may provide a paradigm for a new systems-biology approach in functional genome research aimed at understanding the genetic basis of organ development, physiology and disease.     

Methods for acquisition of quantitative data from confocal images of gene expression in situ

In this review, we summarize original methods for the extraction of quantitative information from confocal images of gene-expression patterns. These methods include image segmentation, the extraction of quantitative numerical data on gene expression, and the removal of background signal and spatial registration. Finally, it is possible to construct a spatiotemporal atlas of gene expression from individual images recorded at each developmental stage. Initially all methods were developed to extract quantitative numerical information from confocal images of segmentation gene expression in Drosophila melanogaster. The application of these methods to Drosophila images makes it possible to reveal new mechanisms in the formation of segmentation gene expression domains, as well as to construct a quantitative atlas of segmentation gene expression. Most image processing procedures can be easily adapted to process a wide range of biological images.     

Microdissection of the gene expression codes driving nephrogenesis

The kidney represents an excellent model system for learning the principles of organogenesis. It is intermediate in complexity, and employs many commonly used developmental processes. As such, kidney development has been the subject of intensive study, using a variety of techniques, including in situ hybridization, organ culture and gene targeting, revealing many critical genes and pathways. Nevertheless, proper organogenesis requires precise patterns of cell type specific differential gene expression, involving very large numbers of genes. This review is focused on the use of global profiling technologies to create an atlas of gene expression codes driving development of different mammalian kidney compartments. Such an atlas allows one to select a gene of interest, and to determine its expression level in each element of the developing kidney, or to select a structure of interest, such as the renal vesicle, and to examine its complete gene expression state. Novel component specific molecular markers are identified, and the changing waves of gene expression that drive nephrogenesis are defined. As the tools continue to improve for the purification of specific cell types and expression profiling of even individual cells it is possible to predict an atlas of gene expression during kidney development that extends to single cell resolution.     

Microdissection of the gene expression codes driving nephrogenesis

The kidney represents an excellent model system for learning the principles of organogenesis. It is intermediate in complexity, and employs many commonly used developmental processes. As such, kidney development has been the subject of intensive study, using a variety of techniques, including in situ hybridization, organ culture and gene targeting, revealing many critical genes and pathways. Nevertheless, proper organogenesis requires precise patterns of cell type specific differential gene expression, involving very large numbers of genes. This review is focused on the use of global profiling technologies to create an atlas of gene expression codes driving development of different mammalian kidney compartments. Such an atlas allows one to select a gene of interest, and to determine its expression level in each element of the developing kidney, or to select a structure of interest, such as the renal vesicle, and to examine its complete gene expression state. Novel component specific molecular markers are identified, and the changing waves of gene expression that drive nephrogenesis are defined. As the tools continue to improve for the purification of specific cell types and expression profiling of even individual cells it is possible to predict an atlas of gene expression during kidney development that extends to single cell resolution.     

The European Renal Genome Project: An Integrated Approach Towards Understanding the Genetics of Kidney Development and Disease

Rapid progress in genome research creates a wealth of information on the functional annotation of mammalian genome sequences. However, as we accumulate large amounts of scientific information we are facing problems of how to integrate and relate the data produced by various genomic approaches. Here, we propose the novel concept of an organ atlas where diverse data from expression maps to histological findings to mutant phenotypes can be queried, compared and visualized in the context of a three-dimensional reconstruction of the organ. We will seek proof of concept for the organ atlas by elucidating genetic pathways involved in development and pathophysiology of the kidney. Such a kidney atlas may provide a paradigm for a new systems-biology approach in functional genome research aimed at understanding the genetic bases of organ development, physiology and disease.Key Words: EuReGene, kidney, genome, development, pathophysiology, genetics     

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 wholemount 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.     

Creation of a three-dimensional atlas by interpolation from Schaltenbrand-Bailey's atlas

A 0.5-mm step atlas was interpolated from Schaltenbrand-Bailey's atlas. By serial display of the atlas with the desired rotation on a three-dimensional axis, a three-dimensional atlas was created. The newly created atlas can be used for computer-processed stereotactic data display. Any three-dimensionally located point can be displayed within 0.25 mm distance from the point, and spatial understanding of anatomical structures and stereotactic data is facilitated by the three-dimensional display.     

PEATmoss (Physcomitrella Expression Atlas Tool): a unified gene expression atlas for the model plant Physcomitrella patens

Physcomitrella patens is a bryophyte model plant that is often used to study plant evolution and development. Its resources are of great importance for comparative genomics and evo‐devo approaches. However, expression data from Physcomitrella patens were so far generated using different gene annotation versions and three different platforms: CombiMatrix and NimbleGen expression microarrays and RNA sequencing. The currently available P. patens expression data are distributed across three tools with different visualization methods to access the data. Here, we introduce an interactive expression atlas, Physcomitrella Expression Atlas Tool (PEATmoss), that unifies publicly available expression data for P. patens and provides multiple visualization methods to query the data in a single web‐based tool. Moreover, PEATmoss includes 35 expression experiments not previously available in any other expression atlas. To facilitate gene expression queries across different gene annotation versions, and to access P. patens annotations and related resources, a lookup database and web tool linked to PEATmoss was implemented. PEATmoss can be accessed at https://peatmoss.online.uni-marburg.de     

A Diffusion-Tensor-Based White Matter Atlas for Rhesus Macaques

Atlases of key white matter (WM) structures in humans are widely available, and are very useful for region of interest (ROI)-based analyses of WM properties. There are histology-based atlases of cortical areas in the rhesus macaque, but none currently of specific WM structures. Since ROI-based analysis of WM pathways is also useful in studies using rhesus diffusion tensor imaging (DTI) data, we have here created an atlas based on a publicly available DTI-based template of young rhesus macaques. The atlas was constructed to mimic the structure of an existing human atlas that is widely used, making results translatable between species. Parcellations were carefully hand-drawn on a principle-direction color-coded fractional anisotropy image of the population template. The resulting atlas can be used as a reference to which registration of individual rhesus data can be performed for the purpose of white-matter parcellation. Alternatively, specific ROIs from the atlas may be warped into individual space to be used in ROI-based group analyses. This atlas will be made publicly available so that it may be used as a resource for DTI studies of rhesus macaques.     

Spatial models to account for variation in observer effort in bird atlases

To assess the importance of variation in observer effort between and within bird atlas projects and demonstrate the use of relatively simple conditional autoregressive (CAR) models for analyzing grid‐based atlas data with varying effort. Pennsylvania and West Virginia, United States of America. We used varying proportions of randomly selected training data to assess whether variations in observer effort can be accounted for using CAR models and whether such models would still be useful for atlases with incomplete data. We then evaluated whether the application of these models influenced our assessment of distribution change between two atlas projects separated by twenty years (Pennsylvania), and tested our modeling methodology on a state bird atlas with incomplete coverage (West Virginia). Conditional Autoregressive models which included observer effort and landscape covariates were able to make robust predictions of species distributions in cases of sparse data coverage. Further, we found that CAR models without landscape covariates performed favorably. These models also account for variation in observer effort between atlas projects and can have a profound effect on the overall assessment of distribution change. Accounting for variation in observer effort in atlas projects is critically important. CAR models provide a useful modeling framework for accounting for variation in observer effort in bird atlas data because they are relatively simple to apply, and quick to run.     

Hypertext atlas of fetal and neonatal pathology

Hypertext atlas of fetal and neonatal pathology is a free resource for pregraduate students of medicine, pathologists and other health professionals dealing with prenatal medicine. The atlas can be found at http://www.muni.cz/atlases. The access is restricted to registered users. Concise texts summarize the gross and microscopic pathology, etiology, and clinical signs of both common and rare fetal and neonatal conditions. The texts are illustrated with over 300 images that are accompanied by short comments. The atlas offers histological pictures of high quality. Virtual microscope interface is used to access the high-resolution histological images. Fetal ultrasound video clips are included. Case studies integrate clinical history, prenatal ultrasonographic examination, gross pathology and histological features. The atlas is available in English (and Czech) and equipped with an active index. The atlas is suitable both for medical students and pathologists as a teaching and reference tool. The atlas is going to be further expanded while keeping the high quality of the images.     

Pipeline for acquisition of quantitative data on segmentation gene expression from confocal images

We describe a data pipeline developed to extract the quantitative data on segmentation gene expression from confocal images of gene expression patterns in Drosophila. The pipeline consists of 5 steps: image segmentation, background removal, temporal characterization of an embryo, data registration and data averaging. This pipeline was successfully applied to obtain quantitative gene expression data at cellular resolution in space and at the 6.5 minute resolution in time, as well as to construct a spatiotemporal atlas of segmentation gene expression. Each data pipeline step can be easily adapted to process a wide range of images of gene expression patterns.     

Pipeline for acquisition of quantitative data on segmentation gene expression from confocal images

We describe a data pipeline developed to extract the quantitative data on segmentation gene expression from confocal images of gene expression patterns in Drosophila. The pipeline consists of five steps: image segmentation, background removal, temporal characterization of an embryo, data registration and data averaging. This pipeline was successfully applied to obtain quantitative gene expression data at cellular resolution in space and at the 6.5-minute resolution in time, as well as to construct a spatiotemporal atlas of segmentation gene expression. Each data pipeline step can be easily adapted to process a wide range of images of gene expression patterns.