We describe a system that permits the automated analysis of reporter gene expression in Caenorhabditis elegans with cellular resolution continuously during embryogenesis. We demonstrate its utility by defining the expression patterns of reporters for several embryonically expressed transcription factors. The invariant cell lineage permits the automated alignment of multiple expression profiles, allowing direct comparison of the expression of different genes' reporters. We also used this system to monitor perturbations to normal development involving changes both in cell-division timing and in cell fate. Systematic application of this system could reveal the gene activity of each cell throughout development. A major goal of current biological research is to understand how the genome directs the process by which a single-cell zygote gives rise to the complexity of a multicellular organism and in turn a new zygote. Simply knowing the full complement of transcriptionally active genes for each cell throughout development would be a major advance and would provide the molecular framework for understanding the network of interactions with which development proceeds. For example, the description of the expression pattern of pair-rule genes such as even skipped in Drosophila melanogaster with high spatial and temporal resolution had been critical in understanding how these genes can cause expressing cells to adopt a fate different from adjacent nonreporter-expressing cells 1 . Several different methods have attempted to capture expression information with high temporal and spatial resolution on a broad scale. Microarray experiments have provided valuable information such as temporal expression in whole embryos or single-time-point expression for specific cell types 2-7 . However, microarray-based analysis is unwieldy and technically challenging when trying to achieve both cellular and high temporal resolution. Imaging an organism more readily provides single-cell resolution of expression by using a transgenic reporter with a visualizable output, or by directly labeling transcripts or proteins. However, typically only one or a few genes are assayed at once, often only at specific times. Integrating such datasets across multiple genes and multiple specimens throughout development generally requires expert anatomists, and expression profiles of individual cells are often lost. The nematode C. elegans presents the possibility of new approaches to the comprehensive description of expression patterns. Because it is transparent from the zygote to the adult and has just 959 somatic cells as an adult, every cell can be visualized throughout the life cycle in living worms. Because a worm develops through an invariant cell lineage, knowledge of an imaged worm's lineage allows unambiguous assignment of the identity of each cell 8 . This potentially allows the alignment of expression patterns from individual worms onto a reference lineage, providing an integrated view of gene expression for each cell. To exploit these possibilities systematically we had previously developed methods that allow automated computational tracing of the C. elegans lineage 9-11 . These methods use custom software to identify nuclei and track them over time, in three-dimensional (3D) movies of worm embryos ubiquitously expressing nuclearlocalized histone-GFP fusion proteins. This system generates highly accurate lineages through the 350-cell stage, which comprises all but the last round of embryonic cell division. Here we extend the system to provide detailed spatiotemporal characterization of reporter gene expression in both wild-type and genetically perturbed embryos. RESULTS To monitor gene expression we generated promoter-histone genefluorescent protein gene reporter constructs, using a gene's 5¢ intergenic sequence to drive expression of an mCherry 12,13 or a DsRed reporter (Table 1) . Fusing the reporter to a C. elegans histone coding sequence directs the reporter to the nucleus to facilitate quantification and assignment of the signal to specific cells. These constructs, although lacking post-transcriptional control signals and perhaps even some transcriptional controls, should drive spatiotemporal expression of the reporter and provide a test of our ability to describe expression patterns over time in single cells. We used microparticle bombardment to generate stably integrated transgenes, to avoid high copy number, mosaicism,
Murray et al. REF have developed a system to automatically trace cell lineage during embryogenesis of C. elegans.
Automated analysis of embryonic gene expression with cellular resolution in C. elegans
Nature Methods
Nature Methods
Biology Medicine