Axel Visel

GenePaint.org: an atlas of gene expression patterns in the mouse embryo

High-throughput instruments were recently developed to determine gene expression patterns on tissue sections by RNA in situ hybridization. The resulting images of gene expression patterns, chiefly of E14.5 mouse embryos, are accessible to the public at http://www.genepaint.org. This relational database is searchable for gene identifiers and RNA probe sequences. Moreover, patterns and intensity of expression in approximately 100 different embryonic tissues are annotated and can be searched using a standardized catalog of anatomical structures. A virtual microscope tool, the Zoom Image Server, was implemented in GenePaint.org and permits interactive zooming and panning across approximately 15,000 high-resolution images.

Comprehensive expression atlas of fibroblast growth factors and their receptors generated by a novel robotic in situ hybridization platform

A recently developed robotic platform termed “Genepaint” can carry out large‐scale nonradioactive in situ hybridization (ISH) on tissue sections. We report a series of experiments that validate this novel platform. Signal‐to‐noise ratio and mRNA detection limits were comparable to traditional ISH procedures, and hybridization was transcript‐specific, even in cases in which probes could have hybridized to several transcripts of a multigene family. We established an atlas of expression patterns of fibroblast growth factors (Fgfs) and their receptors (Fgfrs) for the embryonic day 14.5 mouse embryo. This atlas provides a comprehensive overview of previously known as well as novel sites of expression for this important family of signaling molecules. The Fgf/Fgfr atlas was integrated into the transcriptome database (www.genepaint.org), where individual Fgf and Fgfr expression patterns can be interactively viewed at cellular resolution and where sites of expressions can be retrieved using an anatomy‐based search. Developmental Dynamics 234:371–386, 2005.

Scaffolding by ERK3 regulates MK5 in development

Extracellular‐regulated kinase 3 (ERK3, MAPK6) is an atypical member of the ERKs, lacking the threonine and tyrosine residues in the activation loop, carrying a unique C‐terminal extension and being mainly regulated by its own protein stability and/or by autophosphorylation. Here we show that ERK3 specifically interacts with the MAPK‐activated protein kinase 5 (MK5 or PRAK) in vitro and in vivo. Expression of ERK3 in mammalian cells leads to nuclear‐cytoplasmic translocation and activation of MK5 and to phosphorylation of both ERK3 and MK5. Remarkably, activation of MK5 is independent of ERK3 enzymatic activity, but depends on its own catalytic activity as well as on a region in the C‐terminal extension of ERK3. In mouse embryonic development, mRNA expression patterns of ERK3 and MK5 suggest spatiotemporal coexpression of both kinases. Deletion of MK5 leads to strong reduction of ERK3 protein levels and embryonic lethality at about stage E11, where ERK3 expression in wild‐type mice is maximum, indicating a role of this signalling module in development.

Regulatory Pathway Analysis by High-Throughput In Situ Hybridization

Automated in situ hybridization enables the construction of comprehensive atlases of gene expression patterns in mammals. Such atlases can become Web-searchable digital expression maps of individual genes and thus offer an entryway to elucidate genetic interactions and signaling pathways. Towards this end, an atlas housing ∼1,000 spatial gene expression patterns of the midgestation mouse embryo was generated. Patterns were textually annotated using a controlled vocabulary comprising >90 anatomical features. Hierarchical clustering of annotations was carried out using distance scores calculated from the similarity between pairs of patterns across all anatomical structures. This process ordered hundreds of complex expression patterns into a matrix that reflects the embryonic architecture and the relatedness of patterns of expression. Clustering yielded 12 distinct groups of expression patterns. Because of the similarity of expression patterns within a group, members of each group may be components of regulatory cascades. We focused on the group containing Pax6, an evolutionary conserved transcriptional master mediator of development. Seventeen of the 82 genes in this group showed a change of expression in the developing neocortex of Pax6-deficient embryos. Electromobility shift assays were used to test for the presence of Pax6-paired domain binding sites. This led to the identification of 12 genes not previously known as potential targets of Pax6 regulation. These findings suggest that cluster analysis of annotated gene expression patterns obtained by automated in situ hybridization is a novel approach for identifying components of signaling cascades.

Comprehensive analysis of the expression patterns of the adenylate cyclase gene family in the developing and adult mouse brain.

Adenylate cyclases (Adcys) are components of several developmentally, neurophysiologically, and pharmacologically relevant signaling pathways. A prominent feature of Adcys is their ability to integrate multiple signaling pathways into a single second messenger pathway, the production of cAMP. Nine isoforms of membrane‐bound Adcys are known, each encoded by a distinct gene. These isoforms differ in their response to regulatory upstream pathways as well as in their distribution in the brain and elsewhere. Use of various detection methods and animal species has, however, hampered a direct comparison of expression patterns, so the potential contribution of single isoforms to Adcy activity in different brain regions remains unclear. We have determined the expression patterns of all nine Adcy genes in the embryonic, postnatal day 7, and adult mouse brain by nonradioactive robotic in situ hybridization (ISH). Here we describe the salient features of these patterns. Regional colocalization of Adcy transcripts encoding isoforms with different regulatory properties was detected in the cortex, subregions of the hippocampus, olfactory bulb, thalamus, and striatum. Hence, our expression data support models for modulation of cAMP signaling by combinatorial action of multiple Adcy isoforms. However, in several instances, the expression domains of genes encoding isoforms with similar regulatory properties spatially exclude each other, which is most evident in not previously described expression domains of the embryonic midbrain roof. This is suggestive of functional specialization. J. Comp. Neurol. 496:684–697, 2006.

Enhancer redundancy provides phenotypic robustness in mammalian development

Distant-acting tissue-specific enhancers, which regulate gene expression, vastly outnumber protein-coding genes in mammalian genomes, but the functional importance of this regulatory complexity remains unclear. Here we show that the pervasive presence of multiple enhancers with similar activities near the same gene confers phenotypic robustness to loss-of-function mutations in individual enhancers. We used genome editing to create 23 mouse deletion lines and inter-crosses, including both single and combinatorial enhancer deletions at seven distinct loci required for limb development. Unexpectedly, none of the ten deletions of individual enhancers caused noticeable changes in limb morphology. By contrast, the removal of pairs of limb enhancers near the same gene resulted in discernible phenotypes, indicating that enhancers function redundantly in establishing normal morphology. In a genetic background sensitized by reduced baseline expression of the target gene, even single enhancer deletions caused limb abnormalities, suggesting that functional redundancy is conferred by additive effects of enhancers on gene expression levels. A genome-wide analysis integrating epigenomic and transcriptomic data from 29 developmental mouse tissues revealed that mammalian genes are very commonly associated with multiple enhancers that have similar spatiotemporal activity. Systematic exploration of three representative developmental structures (limb, brain and heart) uncovered more than one thousand cases in which five or more enhancers with redundant activity patterns were found near the same gene. Together, our data indicate that enhancer redundancy is a remarkably widespread feature of mammalian genomes that provides an effective regulatory buffer to prevent deleterious phenotypic consequences upon the loss of individual enhancers.

Adenylate Cyclase 1 dependent refinement of retinotopic maps in the mouse.

Development of the retino-collicular pathway has served as an important model system for examining the cellular mechanisms responsible for the establishment of neuronal maps of the sensory periphery. A consensus has emerged that molecular or chemical cues are responsible for the initial establishment of gross topography in this map, and that activity dependent factors sharpen this initial rough topography into precision. However, there is little evidence available concerning the biochemical signaling mechanisms that are responsible for topographic map refinement in the retino-collicular system. Using a combination of anatomical and biochemical techniques in normal and mutant mice, we provide evidence that Ca2+/Calmodulin regulated Adenylate Cyclase 1 (AC1), which is strongly expressed in the superficial layers of the colliculus, is an important downstream signaling agent for activity dependent map refinement in the superior colliculus.

A Gene Expression Map of the Mouse Brain

Tens of thousands of novel genes have been identified by systematic DNA sequencing of a variety of genomes. Such gene sequences create a tremendous opportunity for addressing gene function by a variety of experimental approaches. To be attractive for genome-wide analysis, experimental strategies need not only to be efficient (high-throughput) but results also have to be systematized and made accessible in a way that they can be promptly leveraged by the scientific community. Having a genome-wide atlas of gene expression patterns with cellular resolution would be a rich resource to tackle a broad spectrum of biological and medical questions. The techniques to analyze gene expression have long been invented: mRNA in situ-hybridization (ISH) and antibody-based immunohistochemistry (IC). Traditionally, both ISH and IC are carried out on a gene-by-gene basis. Here we describe instrumentation (termed GenePaint) that permits automation of many aspects of ISH and image data acquisition. The throughput that is achieved renders investigations at a chromosome- or even genome-wide scale highly feasible. To effectively disseminate gene expression data, we have developed a database (www.gene-paint.org). Systematic annotation of gene expression data in reference to a standardized catalogue of anatomical structures makes it possible to compare expression patterns between specimens. For viewing expression patterns, GenePaint.org provides a “virtual microscope” which enables zooming and translocation of expression patterns. GenePaint is currently applied to large-scale studies of gene expression in the CNS of the mouse.