GenePaint is a digital atlas of gene expression patterns in various tissues and species with strong focus on mouse embryos. Expression patterns are determined by non-radioactive in situ hybridization on serial tissue sections.
The database is gene-centric and entries can be searched either by gene name, accession number, sequence homology or site of expression. The website features a "virtual microscope" that enables zooming into images down to cellular resolution.
We acknowledge the contribution of the EURExpress consortium.
To assist in the initial identification of sites of gene expression, maps of sagittal sections at embryonic day 14.5 are available in an interactive anatomy atlas. The E14.5 C57BL/6J embryo used in the atlas was prepared, sectioned and imaged identically to the embryos used for in situ hybridization (see Methods of Data Production). Section thickness is 25µm and inter-section distance is 150µm. Tissue was stained with Thioninacetate (Nissl-method). All sections were digitally scanned using a 5x objective. Structures annotated for gene expression are indicated in the maps with red pointers. Boundaries between brain regions are indicated with dashed yellow lines. Both, the in situ hybridization section after a performed search and the appropriate atlas section can be viewed side-by-side.
Probes were designed and synthesized from freely available cDNA clone resources. For genes not represented in a public collection, templates were produced by PCR from cDNA using gene-specific primers. Templates and hence riboprobes were ∼1000 nt long. Transcript expression was detected with non-radioactive riboprobes tagged with digoxygenin by in vitro-transcription from DNA templates using appropriate primers and RNA polymerases.
Mice were maintained on a 12-h light/dark cycle with light being turned on at 07:00. Animals were anesthetized with isoflurane (Abbott Laboratories) and either killed by decapitation (for brain preparations) or through cervical dislocation (for embryo collection).
were briefly washed in cold phosphate-buffered saline, blotted dry with a filter paper and transferred into ice-cold OCT 4583 (Tissue-Tek, Sakura). Tissues were transferred to a freezing chamber (Figure 1) within 5 min after dissection. The custom-made freezing chamber consists of a square copper base and transparent Plexiglass side walls. The chamber was placed on a specially designed, movable stage fitted to a stereomicroscope. Through translation and rotation of this stage, the edges of the chamber were aligned parallel to a grid placed into one of the eyepieces of the stereomicroscope. The chamber was filled with OCT at room temperature and the tissue was submerged in OCT and oriented appropriately with the aid of a blunt dissecting needle. Orientation of the embryo was correct if the sagittal mid-plane of the embryo was parallel to a side wall of the chamber as judged by viewing the specimen frontally through the transparent side walls. In addition, the dorsal midline of the embryo had to be parallel to the gridlines of the eyepiece. In the case of brain the sagittal mid-plane was oriented parallel to the side walls. After orientation, usually within 1 min, the chamber containing the specimen is placed on the aluminum block immersed in ethanol contained in a cooler that was kept in a -80° C freezer. Alternatively, the freezing chamber was placed on a block of dry ice. Once the OCT was frozen up to the level of the specimen, the chamber was put onto the freezing shelf of a cryostat for a few hours. Thereafter, tissue axis and specimen number was written on the block, the chamber was disassembled and the blocks were placed into plastic bags and stored at –80°C for several months and even years.
At least one day prior to sectioning, OCT blocks were transferred to a –20°C freezer and then mounted on a cryostat chuck. Because of the orthogonal walls of the O.C.T. block, specimens can be accurately oriented. The chuck is fastened to the goniometer of the cryostat and oriented so that the edge of the knife cuts parallel to one of the cardinal planes of the specimen. A Leica cryostat (Model CM 3050S) is used and sections are 20µm or 25µm thick. Superfrost slides are placed into aluminum slide holders which permits precise positioning of sections onto the slides.
Following sectioning, slides are kept in slide racks in the cryostat until ready for fixation. Fixation, carried out in a Leica Autostainer XL, is in 4% paraformaldehyde for 20 min at room temperature. Subsequently, slides are washed for 5 min in PBS, acetylated and dried by passing them through an ethanol series ending with 100 % ethanol. After drying at 30°C, slides are stored at –80°C in boxes containing a drying agent and sealed with electrical tape.
To increase sensitivity of in situ hybridization, tyramide-biotin amplification was used (also known as catalyzed reporter deposition; Speel, E.J. [1999]. Histochem Cell. Biol. 112, 89-113). Following hybridization of cellular mRNA with digoxigenin-11-dUTP-tagged RNA, peroxidase-coupled anti-digoxygenin antibody was added. The peroxidase moiety of the antibody activates a biotin-tyramide conjugate that covalent attaches biotin to proteins in the vicinity of the riboprobe. Biotin was subsequently detected by an alkaline phosphatase-based BCIP/NPT color reaction resulting in a precipitate covering the cell expressing the gene in question.
The slides carrying tissue sections were integrated into flow-through chamber (Figure 3), 48 of which are accommodated in a thermo rack located on a Tecan Genesis™ (or Freedom Evo™) liquid handling platform (Figure 4). Depending on the platform size, up to 4 thermo racks can be used. Turnaround time for a single run with 4 racks is ~24 h.
Slides are coverslipped with a water-based medium and photographed in a bright field or DIC light microscope equipped with a motorized stage that moves the slide in front of the objective. The equipment used consists of a Leica DM-RXA2 microscope, a motorized Märzhäuser stage that accommodates up to eight slides, a Leica electronic focusing system, a Hitachi HV-C20A CCD color camera and a PC based custom-made controller that drives and coordinates stage and camera (Figure 5). Embryo sections are too large to be photographed as a whole. Therefore, the motorized stage moves the sections in a stepwise mosaic fashion in front of the objective and at each step an image is taken. Subsequently images are stitched together, cropped, saved as TIFF file and uploaded on the GenePaint database. Most images were captured with a 10 x objective in which case the numerical aperture of the objective lens was 0.4 and the pixel size was 1.6 µm/px. At an early phase of the GenePaint project, images were collected with a 5 x objective (numerical aperture 0.15) and 3.2 µm/px.
Expression patterns revealed by in situ hybridization were annotated by experts and volunteers. Emphasis was placed on those genes that exhibited regional expression. Expression strength and expression pattern for each anatomical structure were scored (see below) and entered into hierarchically organized collapsible and extendable tree of organs/tissues (Figure 6, top). For easier recognition an icon for strength and pattern was generated ant placed at the left margin (Figure 6, bottom). Annotation enables to query GenePaint.org for gene expression in particular anatomical regions.
Cartilage may occasionally display weak non-specific signal (Figure 8A). The myocardium of the embryonic heart can exhibit splotch-like round stains of uniform intensity that can readily be distinguished from the granular precipitate characteristic for true signal (Figure 8B). Single sections within a data set may exhibit streaks or smears across the whole specimen, usually resulting either from sectioning problems or from uneven exposure to reagents during in-situ hybridization (Figure 8C, D). These artifacts are easily identified because the stained regions do not agree with anatomical boundaries and are absent on adjacent sections.
Stage | Strain | Type of Specimen | Sections per Set | Position of Section Shown |
E10.5 | NMRI | whole embryo | 1-5 | representative section |
E14.5 | NMRI | whole embryo | 24 | 100-175µm constant spacing |
E15.5 | NMRI | head | 10 | selected standard planes (see atlas) |
P7 | C57BL/6 | brain | 11 | selected standard planes (see atlas) |
P56 | C57BL/6 | brain | 10 | selected standard planes (see atlas) |
This mouse metabolism database was designed to navigate complex metabolic maps with a keen eye on the gene expression landscapes of enzymes and transporters.
http://www.metscout.mpg.de/Funded by the EU this project was the first to achieve the transcriptome-wide acquisition of gene expression patterns by ISH in the mouse embryo.
http://www.eurexpress.org/ee/The goal of this EU project was to discover genes responsible for renal development and disease. The original ISH-Atlas generated for EuReGene is now part of GUDMAP .
A molecular map showing where all genes are expressed in all regions of the mouse brain.
http://brain-map.org .