A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

Prediction of alternative isoforms from exon expression levels in RNA-Seq experiments.

Alternative splicing, polyadenylation of pre-messenger RNA molecules and differential promoter usage can produce a variety of transcript isoforms whose respective expression levels are regulated in time and space, thus contributing specific biological functions. However, the repertoire of mammalian alternative transcripts and their regulation are still poorly understood. Second-generation sequencing is now opening unprecedented routes to address the analysis of entire transcriptomes. Here, we developed methods that allow the prediction and quantification of alternative isoforms derived solely from exon expression levels in RNA-Seq data. These are based on an explicit statistical model and enable the prediction of alternative isoforms within or between conditions using any known gene annotation, as well as the relative quantification of known transcript structures. Applying these methods to a human RNA-Seq dataset, we validated a significant fraction of the predictions by RT-PCR. Data further showed that these predictions correlated well with information originating from junction reads. A direct comparison with exon arrays indicated improved performances of RNA-Seq over microarrays in the prediction of skipped exons. Altogether, the set of methods presented here comprehensively addresses multiple aspects of alternative isoform analysis. The software is available as an open-source R-package called Solas at http://cmb.molgen.mpg.de/2ndGenerationSequencing/Solas/.

Subapical localization of the dopamine D3 receptor in proximal tubules of the rat kidney.

The dopamine D3 receptor (D3R), intensively studied in neuroscience, also plays an important role in the regulation of renal and cardiovascular function. In contrast to functional findings, less information is available on its localization in the kidney. Neither RT-PCR studies nor radioligand binding assays are suitable to selectively determine the distribution of renal D3R at the level of cellular or even subcellular structures. We studied the renal D3R distribution in Sprague-Dawley rats by a polyclonal antiserum directed against an epitope in the third intracytoplasmic loop. D3R immunoreactivity was detected by indirect immunofluorescence and confocal laser scanning microscopy. D3R staining was confined to the renal cortex and occurred in proximal convoluted tubules near or in direct connection with the urinary pole of the glomeruli. The fluorescent spots were restricted to the subapical portion of the proximal tubular cells. Double staining with the F-actin marker phalloidin revealed a localization of the D3R below the brush border region. However, staining by anti-beta1/beta2-adaptins, recognizing clathrin-coated compartments, did not correspond to the distribution of the D3R signal. This is the first description of a D3R accumulation in a cytoplasmic pool in the kidney, probably corresponding to a recycling mechanism or storage compartment.