Using DNA sequences 5′ to open reading frames, we have constructed green fluorescent protein (GFP) fusions and generated spatial and temporal tissue expression profiles for 1,886 specific genes in the nematode Caenorhabditis elegans. This effort encompasses about 10% of all genes identified in this organism. GFP-expressing wild-type animals were analyzed at each stage of development from embryo to adult. We have identified 5′ DNA regions regulating expression at all developmental stages and in 38 different cell and tissue types in this organism. Among the regulatory regions identified are sequences that regulate expression in all cells, in specific tissues, in combinations of tissues, and in single cells. Most of the genes we have examined in C. elegans have human orthologs. All the images and expression pattern data generated by this project are available at WormAtlas (http://gfpweb.aecom.yu.edu/index) and through WormBase (http://www.wormbase.org).

Differential regulation of gene expression is essential for cell fate specification in metazoans. Characterizing the transcriptional activity of gene promoters, in time and in space, is therefore a critical step toward understanding complex biological systems. Here we present an in vivo spatiotemporal analysis for approximately 900 predicted C. elegans promoters (approximately 5% of the predicted protein-coding genes), each driving the expression of green fluorescent protein (GFP). Using a flow-cytometer adapted for nematode profiling, we generated 'chronograms', two-dimensional representations of fluorescence intensity along the body axis and throughout development from early larvae to adults. Automated comparison and clustering of the obtained in vivo expression patterns show that genes coexpressed in space and time tend to belong to common functional categories. Moreover, integration of this data set with C. elegans protein-protein interactome data sets enables prediction of anatomical and temporal interaction territories between protein partners.

The nematode Caenorhabditis elegans is a powerful model system to study contemporary biological problems. This system would be even more useful if we had mutations in all the genes of this multicellular metazoan. The combined efforts of the C. elegans Deletion Mutant Consortium and individuals within the worm community are moving us ever closer to this goal. At present, of the 20,377 protein-coding genes in this organism, 6764 genes with associated molecular lesions are either deletions or null mutations (WormBase WS220). Our three laboratories have contributed the majority of mutated genes, 6841 mutations in 6013 genes. The principal method we used to detect deletion mutations in the nematode utilizes polymerase chain reaction (PCR). More recently, we have used array comparative genome hybridization (aCGH) to detect deletions across the entire coding part of the genome and massively parallel short-read sequencing to identify nonsense, splicing, and missense defects in open reading frames. As deletion strains can be frozen and then thawed when needed, these strains will be an enduring community resource. Our combined molecular screening strategies have improved the overall throughput of our gene-knockout facilities and have broadened the types of mutations that we and others can identify. These multiple strategies should enable us to eventually identify a mutation in every gene in this multicellular organism. This knowledge will usher in a new age of metazoan genetics in which the contribution to any biological process can be assessed for all genes.

The current Caenorhabditis elegans genomic annotation has many genes organized in operons. Using directionally stitched promoterϻGFP methodology, we have conducted the largest survey to date on the regulatory regions of annotated C. elegans operons and identified 65, over 25% of those studied, with internal promoters. We have termed these operons "hybrid operons." GFP expression patterns driven from internal promoters differ in tissue specificity from expression of operon promoters, and serial analysis of gene expression data reveals that there is a lack of expression correlation between genes in many hybrid operons. The average length of intergenic regions with putative promoter activity in hybrid operons is larger than previous estimates for operons as a whole. Genes with internal promoters are more commonly involved in gene duplications and have a significantly lower incidence of alternative splicing than genes without internal promoters, although we have observed almost all trans-splicing patterns in these two distinct groups. Finally, internal promoter constructs are able to rescue lethal knockout phenotypes, demonstrating their necessity in gene regulation and survival. Our work suggests that hybrid operons are common in the C. elegans genome and that internal promoters influence not only gene organization and expression but also operon evolution.