Rearing C. elegans on Parafilm-wrapped NGM Plates Impacts Habituation Behavior.

Scientists use Parafilm to seal Caenorhabditis elegans cultures on Nematode Growth Media (NGM) petri plates for short-term storage to reduce the likelihood of contamination and improve moisture retention. However, we found that maintaining worms on plates wrapped with Parafilm can affect multiple behavioral metrics when assaying tap-habituation behavior using the Multi-Worm Tracker (MWT). Most notably, worms cultured on parafilm-wrapped NGM plates exhibited slower speed of initial response to tap followed by marked sensitization. These findings suggest that labs should be conscious of the possibility that Parafilm may induce behavioral changes in C. elegans when conducting experiments.

Neuroligin Plays a Role in Ethanol-Induced Disruption of Memory and Corresponding Modulation of Glutamate Receptor Expression.

Exposure to alcohol causes deficits in long-term memory formation across species. Using a long-term habituation memory assay in Caenorhabditis elegans, the effects of ethanol on long-term memory (> 24 h) for habituation were investigated. An impairment in long-term memory was observed when animals were trained in the presence of ethanol. Cues of internal state or training context during testing did not restore memory. Ethanol exposure during training also interfered with the downregulation of AMPA/KA-type glutamate receptor subunit (GLR-1) punctal expression previously associated with long-term memory for habituation in C. elegans. Interestingly, ethanol exposure alone had the opposite effect, increasing GLR-1::GFP punctal expression. Worms with a mutation in the C. elegans ortholog of vertebrate neuroligins (nlg-1) were resistant to the effects of ethanol on memory, as they displayed both GLR-1::GFP downregulation and long-term memory for habituation after training in the presence of ethanol. These findings provide insights into the molecular mechanisms through which alcohol consumption impacts memory.

The contribution of C. elegans neurogenetics to understanding neurodegenerative diseases.

Since Caenorhabditis elegans was first introduced as a genetic model organism by Sydney Brenner, researchers studying it have made significant contributions in numerous fields including investigations of the pathophysiology of neurodegenerative diseases. The simple anatomy, optical transparency, and short life-span of this small nematode together with the development and curation of many openly shared resources (including the entire genome, cell lineage and the neural map of the animal) allow researchers using C. elegans to move their research forward rapidly in an immensely collaborative community. These resources have allowed researchers to use C. elegans to study the cellular processes that may underlie human diseases. Indeed, many disease-associated genes have orthologs in C. elegans, allowing the effects of mutations in these genes to be studied in relevant and reproducible neuronal cell-types at single-cell resolution in vivo. Here we review studies that have attempted to establish genetic models of specific human neurodegenerative diseases (ALS, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease) in C. elegans and what they have contributed to understanding the molecular and genetic underpinnings of each disease. With continuous advances in genome engineering, research conducted using this small organism first established by Brenner, Sulston and their contemporaries will continue to contribute to the understanding of human nervous diseases.

Systematic phenomics analysis of autism-associated genes reveals parallel networks underlying reversible impairments in habituation.

A major challenge facing the genetics of autism spectrum disorders (ASDs) is the large and growing number of candidate risk genes and gene variants of unknown functional significance. Here, we used Caenorhabditis elegans to systematically functionally characterize ASD-associated genes in vivo. Using our custom machine vision system, we quantified 26 phenotypes spanning morphology, locomotion, tactile sensitivity, and habituation learning in 135 strains each carrying a mutation in an ortholog of an ASD-associated gene. We identified hundreds of genotype-phenotype relationships ranging from severe developmental delays and uncoordinated movement to subtle deficits in sensory and learning behaviors. We clustered genes by similarity in phenomic profiles and used epistasis analysis to discover parallel networks centered on CHD8•chd-7 and NLGN3•nlg-1 that underlie mechanosensory hyperresponsivity and impaired habituation learning. We then leveraged our data for in vivo functional assays to gauge missense variant effect. Expression of wild-type NLG-1 in nlg-1 mutant C. elegans rescued their sensory and learning impairments. Testing the rescuing ability of conserved ASD-associated neuroligin variants revealed varied partial loss of function despite proper subcellular localization. Finally, we used CRISPR-Cas9 auxin-inducible degradation to determine that phenotypic abnormalities caused by developmental loss of NLG-1 can be reversed by adult expression. This work charts the phenotypic landscape of ASD-associated genes, offers in vivo variant functional assays, and potential therapeutic targets for ASD.

CRISPR-Cas9 human gene replacement and phenomic characterization in Caenorhabditis elegans to understand the functional conservation of human genes and decipher variants of uncertain significance.

Our ability to sequence genomes has vastly surpassed our ability to interpret the genetic variation we discover. This presents a major challenge in the clinical setting, where the recent application of whole-exome and whole-genome sequencing has uncovered thousands of genetic variants of uncertain significance. Here, we present a strategy for targeted human gene replacement and phenomic characterization, based on CRISPR-Cas9 genome engineering in the genetic model organism Caenorhabditis elegans, that will facilitate assessment of the functional conservation of human genes and structure-function analysis of disease-associated variants with unprecedented precision. We validate our strategy by demonstrating that direct single-copy replacement of the C. elegans ortholog (daf-18) with the critical human disease-associated gene phosphatase and tensin homolog (PTEN) is sufficient to rescue multiple phenotypic abnormalities caused by complete deletion of daf-18, including complex chemosensory and mechanosensory impairments. In addition, we used our strategy to generate animals harboring a single copy of the known pathogenic lipid phosphatase inactive PTEN variant (PTEN-G129E), and showed that our automated in vivo phenotypic assays could accurately and efficiently classify this missense variant as loss of function. The integrated nature of the human transgenes allows for analysis of both homozygous and heterozygous variants and greatly facilitates high-throughput precision medicine drug screens. By combining genome engineering with rapid and automated phenotypic characterization, our strategy streamlines the identification of novel conserved gene functions in complex sensory and learning phenotypes that can be used as in vivo functional assays to decipher variants of uncertain significance.

An intersectional gene regulatory strategy defines subclass diversity of C. elegans motor neurons.

A core principle of nervous system organization is the diversification of neuron classes into subclasses that share large sets of features but differ in select traits. We describe here a molecular mechanism necessary for motor neurons to acquire subclass-specific traits in the nematode Caenorhabditis elegans. Cholinergic motor neuron classes of the ventral nerve cord can be subdivided into subclasses along the anterior-posterior (A-P) axis based on synaptic connectivity patterns and molecular features. The conserved COE-type terminal selector UNC-3 not only controls the expression of traits shared by all members of a neuron class, but is also required for subclass-specific traits expressed along the A-P axis. UNC-3, which is not regionally restricted, requires region-specific cofactors in the form of Hox proteins to co-activate subclass-specific effector genes in post-mitotic motor neurons. This intersectional gene regulatory principle for neuronal subclass diversification may be conserved from nematodes to mice.