Laura J. Grundy

A database of Caenorhabditis elegans behavioral phenotypes

Using low-cost automated tracking microscopes, we have generated a behavioral database for 305 Caenorhabditis elegans strains, including 76 mutants with no previously described phenotype. The growing database currently consists of 9,203 short videos segmented to extract behavior and morphology features, and these videos and feature data are available online for further analysis. The database also includes summary statistics for 702 measures with statistical comparisons to wild-type controls so that phenotypes can be identified and understood by users.

A dictionary of behavioral motifs reveals clusters of genes affecting Caenorhabditis elegans locomotion

Visible phenotypes based on locomotion and posture have played a critical role in understanding the molecular basis of behavior and development in Caenorhabditis elegans and other model organisms. However, it is not known whether these human-defined features capture the most important aspects of behavior for phenotypic comparison or whether they are sufficient to discover new behaviors. Here we show that four basic shapes, or eigenworms, previously described for wild-type worms, also capture mutant shapes, and that this representation can be used to build a dictionary of repetitive behavioral motifs in an unbiased way. By measuring the distance between each individuals behavior and the elements in the motif dictionary, we create a fingerprint that can be used to compare mutants to wild type and to each other. This analysis has revealed phenotypes not previously detected by real-time observation and has allowed clustering of mutants into related groups. Behavioral motifs provide a compact and intuitive representation of behavioral phenotypes.

Changes in Postural Syntax Characterize Sensory Modulation and Natural Variation of C. elegans Locomotion.

Locomotion is driven by shape changes coordinated by the nervous system through time; thus, enumerating an animals complete repertoire of shape transitions would provide a basis for a comprehensive understanding of locomotor behaviour. Here we introduce a discrete representation of behaviour in the nematode C. elegans. At each point in time, the worm’s posture is approximated by its closest matching template from a set of 90 postures and locomotion is represented as sequences of postures. The frequency distribution of postural sequences is heavy-tailed with a core of frequent behaviours and a much larger set of rarely used behaviours. Responses to optogenetic and environmental stimuli can be quantified as changes in postural syntax: worms show different preferences for different sequences of postures drawn from the same set of templates. A discrete representation of behaviour will enable the use of methods developed for other kinds of discrete data in bioinformatics and language processing to be harnessed for the study of behaviour.

Spatial Asymmetry in the Mechanosensory Phenotypes of the C. elegans DEG/ENaC Gene mec-10

DEG/ENaC channels have been broadly implicated in mechanosensory transduction, yet many questions remain about how these proteins contribute to complexes that sense mechanical stimuli. In C. elegans, two DEG/ENaC channel subunits are thought to contribute to a gentle touch transduction complex: MEC-4, which is essential for gentle touch sensation, and MEC-10, whose importance is less well defined. By characterizing a mec-10 deletion mutant, we have found that MEC-10 is important, but not essential, for gentle touch responses in the body touch neurons ALM, PLM, and PVM. Surprisingly, the requirement for MEC-10 in ALM and PLM is spatially asymmetric; mec-10 animals show significant behavioral and physiological responses to stimulation at the distal end of touch neuron dendrites, but respond poorly to stimuli applied near the neuronal cell body. The subcellular distribution of a rescuing MEC-10::GFP translational fusion was found to be restricted to the neuronal cell body and proximal dendrite, consistent with the hypothesis that MEC-10 protein is asymmetrically distributed within the touch neuron process. These results suggest that MEC-10 may contribute to only a subset of gentle touch mechanosensory complexes found preferentially at the proximal dendrite.

The Bright Fluorescent Protein mNeonGreen Facilitates Protein Expression Analysis In Vivo

The Green Fluorescent Protein (GFP) has been tremendously useful in investigating cell architecture, protein localization, and protein function. Recent developments in transgenesis and genome editing methods now enable working with fewer transgene copies and, consequently, with physiological expression levels. However, lower signal intensity might become a limiting factor. The recently developed mNeonGreen protein is a brighter alternative to GFP in vitro. The goal of the present study was to determine how mNeonGreen performs in vivo in Caenorhabditis elegans—a model used extensively for fluorescence imaging in intact animals. We started with a side-by-side comparison between cytoplasmic forms of mNeonGreen and GFP expressed in the intestine, and in different neurons, of adult animals. While both proteins had similar photostability, mNeonGreen was systematically 3–5 times brighter than GFP. mNeonGreen was also used successfully to trace endogenous proteins, and label specific subcellular compartments such as the nucleus or the plasma membrane. To further demonstrate the utility of mNeonGreen, we tested transcriptional reporters for nine genes with unknown expression patterns. While mNeonGreen and GFP reporters gave overall similar expression patterns, low expression tissues were detected only with mNeonGreen. As a whole, our work establishes mNeonGreen as a brighter alternative to GFP for in vivo imaging in a multicellular organism. Furthermore, the present research illustrates the utility of mNeonGreen to tag proteins, mark subcellular regions, and describe new expression patterns, particularly in tissues with low expression.

PACRG, a protein linked to ciliary motility, mediates cellular signaling

Cilia are cellular projections that can be motile to generate fluid flow or nonmotile to enable signaling. Both forms are based on shared components, and proteins involved in ciliary motility, like PACRG, may also function in ciliary signaling. Caenorhabditis elegans PACRG acts in a subset of nonmotile cilia to influence a learning behavior and promote longevity.

Neuropeptides encoded by nlp-49 modulate locomotion, arousal and egg-laying behaviours in Caenorhabditis elegans via the receptor SEB-3

Neuropeptide signalling has been implicated in a wide variety of biological processes in diverse organisms, from invertebrates to humans. The Caenorhabditis elegans genome has at least 154 neuropeptide precursor genes, encoding over 300 bioactive peptides. These neuromodulators are thought to largely signal beyond ‘wired’ chemical/electrical synapse connections, therefore creating a ‘wireless’ network for neuronal communication. Here, we investigated how behavioural states are affected by neuropeptide signalling through the G protein-coupled receptor SEB-3, which belongs to a bilaterian family of orphan secretin receptors. Using reverse pharmacology, we identified the neuropeptide NLP-49 as a ligand of this evolutionarily conserved neuropeptide receptor. Our findings demonstrate novel roles for NLP-49 and SEB-3 in locomotion, arousal and egg-laying. Specifically, high-content analysis of locomotor behaviour indicates that seb-3 and nlp-49 deletion mutants cause remarkably similar abnormalities in movement dynamics, which are reversed by overexpression of wild-type transgenes. Overexpression of NLP-49 in AVK interneurons leads to heightened locomotor arousal, an effect that is dependent on seb-3. Finally, seb-3 and nlp-49 mutants also show constitutive egg-laying in liquid medium and alter the temporal pattern of egg-laying in similar ways. Together, these results provide in vivo evidence that NLP-49 peptides act through SEB-3 to modulate behaviour, and highlight the importance of neuropeptide signalling in the control of behavioural states. This article is part of a discussion meeting issue ‘Connectome to behaviour: modelling C. elegans at cellular resolution’.

High-Throughput Controlled Mechanical Stimulation and Functional Imaging In Vivo

Understanding mechanosensation and other sensory behavior in genetic model systems such as C. elegans is relevant to many human diseases. These studies conventionally require immobilization by glue and manual delivery of stimuli, leading to low experimental throughput and high variability. Here we present a microfluidic platform that delivers precise mechanical stimuli robustly. The system can be easily used in conjunction with functional imaging and optical interrogation techniques, as well as other capabilities such as sorting or more sophisticated fluid delivery schemes. The platform is fully automated, thereby greatly enhancing the throughput and robustness of experiments. We show that behavior of the well-known gentle and harsh touch neurons and their receptive fields can be recapitulated in our system. Using calcium dynamics as a readout, we demonstrate the ability to perform a drug screen in vivo. Furthermore, using an integrated chip platform that can deliver both mechanical and chemical stimuli, we examine sensory integration in interneurons in response to multimodal sensory inputs. We envision that this system will be able to greatly accelerate the discovery of genes and molecules involved in mechanosensation and multimodal sensory behavior, as well as the discovery of therapeutics for related diseases.