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The Caenorhabditis elegans cell lineage is nearly invariant. Whether this stereotyped cell-division pattern promotes reproducibility in cell shapes/positions is not generally known, as manual spatiotemporal cell-shape/position alignments are labor-intensive, and fully-automated methods are not described. Here, we report automated algorithms for spatiotemporal alignments of C. elegans embryos from pre-morphogenesis to motor-activity initiation. We use sparsely-labeled green-fluorescent nuclei and a pan-nuclear red-fluorescent reporter to register consecutive imaging time points and compare embryos. Using our method, we monitor early assembly of the nerve-ring (NR) brain neuropil. While NR pioneer neurons exhibit reproducible growth kinetics and axon positions, cell-body placements are variable. Thus, pioneer-neuron axon locations, but not cell-body positions, are under selection. We also show that anterior NR displacement in cam-1/ROR Wnt-receptor mutants is not an early NR assembly defect. Our results demonstrate the utility of automated spatiotemporal alignments of C. elegans embryos, and uncover key principles guiding nervous-system development in this animal.

Programmed cell death is a ubiquitous process in metazoan development. Apoptosis, one cell death form, has been studied extensively. However, mutations inactivating key mammalian apoptosis regulators do not block most developmental cell culling, suggesting that other cell death pathways are likely important. Recent work in the nematode Caenorhabditis elegans identified a non-apoptotic cell death form mediating the demise of the male-specific linker cell. This cell death process (LCD, linker cell-type death) is morphologically conserved, and its molecular effectors also mediate axon degeneration in mammals and Drosophila. To develop reagents to manipulate LCD, we established a simple high-throughput screening protocol for interrogating the effects of small molecules on C. elegans linker cell death in vivo. From 23,797 compounds assayed, 11 reproducibly block linker cell death onset. Of these, five induce animal lethality, and six promote a reversible developmental delay. These results provide proof-of principle validation of our screening protocol, demonstrate that developmental progression is required for linker cell death, and suggest that larger scale screens may identify LCD-specific small-molecule regulators that target the LCD execution machinery., Competing Interests: The authors have declared that no competing interests exist.

Many organisms respond to food deprivation by altering their pattern of movement, often in ways that appear to facilitate dispersal. While the behavior of the nematode C. elegans in the presence of attractants has been characterized, long-range movement in the absence of external stimuli has not been examined in this animal. Here we investigate the movement pattern of individual C. elegans over times of ∼1 hour after removal from food, using two custom imaging set-ups that allow us to track animals on large agar surfaces of 22 cm×22 cm. We find that a sizeable fraction of the observed trajectories display directed motion over tens of minutes. Remarkably, this directional persistence is achieved despite a local orientation memory that decays on the scale of about one minute. Furthermore, we find that such trajectories cannot be accounted for by simple random, isotropic models of animal locomotion. This directional behavior requires sensory neurons, but appears to be independent of known sensory signal-transduction pathways. Our results suggest that long-range directional behavior of C. elegans may not be driven by sensory cues.

Background: Highly parallel sequencing technologies have become important tools in the analysis of sequence polymorphisms on a genomic scale. However, the development of customized software to analyze data produced by these methods has lagged behind., Methods/principal Findings: Here I describe a tool, 'galign', designed to identify polymorphisms between sequence reads obtained using Illumina/Solexa technology and a reference genome. The 'galign' alignment tool does not use Smith-Waterman matrices for sequence comparisons. Instead, a simple algorithm comparing parsed sequence reads to parsed reference genome sequences is used. 'galign' output is geared towards immediate user application, displaying polymorphism locations, nucleotide changes, and relevant predicted amino-acid changes for ease of information processing. To do so, 'galign' requires several accessory files easily derived from an annotated reference genome. Direct sequencing as well as in silico studies demonstrate that 'galign' provides lesion predictions comparable in accuracy to available prediction programs, accompanied by greater processing speed and more user-friendly output. We demonstrate the use of 'galign' to identify mutations leading to phenotypic consequences in C. elegans., Conclusion/significance: Our studies suggest that 'galign' is a useful tool for polymorphism discovery, and is of immediate utility for sequence mining in C. elegans.

In genetic screens, the number of mutagenized gametes examined is an important parameter for evaluating screen progress, the number of genes of a given mutable phenotype, gene size, cost, and labor. Since genetic screens often entail examination of thousands or tens of thousands of animals, strategies for optimizing genetics screens are important for minimizing effort while maximizing the number of mutagenized gametes examined. To date, such strategies have not been described for genetic screens in the nematode Caenorhabditis elegans. Here we review general principles of genetic screens in C. elegans, and use a modified binomial strategy to obtain a general expression for the number of mutagenized gametes examined in a genetic screen. We use this expression to calculate optimal screening parameters for a large range of genetic screen types. In addition, we developed a simple online genetic-screen-optimization tool that can be used independently of this paper. Our results demonstrate that choosing the optimal F2-to-F1 screening ratio can significantly improve screen efficiency.