title: The Automatic Tracking Of <i>Caenorhabditis elegans</i> And Its Use In Determining Genetic Function
Creators: name: Grundy, Laura Jayne
Publisher: name: The Open University
Year: 2018

description: Even with its simple nervous system, the nematode worm <i>Caenorhabditis elegans</i> can display a range of complex behaviours. Movement can be viewed as the main output of the <i>C. elegans</i> nervous system, and aberrations in the worm’s locomotion can be used as an indicator for genetic function in mutant strains of <i>C. elegans</i>. Automated tracking of <i>C. elegans</i> locomotion has been used to determine phenotypic fingerprints for ~300 mutant <i>C. elegans</i> strains. Two methods of creating phenotypic fingerprints were used. The first based on pre-determined micro-behaviours previously described in worms, but never before analysed using automated tracking. The second used the tracking data itself to determine micro-motifs, repeated sets of behaviours observed at least twice in at least two mutant or wild-type strains. <br><br> Both methods of clustering successfully grouped together strains with mutations in genes known to interact together, verifying that the technique is able to detect meaningful connections between mutant strains. The following step was to determine whether the technique can be used to establish connections between genes on unknown function. A pair of strains with mutations in DEG/ENaC subunit encoding genes clustered strongly together using the micro-motif method, due to similar defects in their behaviours upon turning. The function of these genes, <i>asic-2</i> and <i>acd-5</i>, was unknown. Upon further investigation it was found that the two genes are expressed in different classes of neurons, the IL2s in the case of <i>asic-2</i> and the ASIs in the case of <i>acd-5</i>. Following investigation into behaviours known to be modulated by these two neuron classes it was found that the mutant strains displayed mutant phenotypes in similar behaviours, but that their mutant phenotypes are opposing. Mutations in <i>asic-2</i> cause increased lifespan and healthspan and a reduction in dauer entry in response to exogenous, purified ascarosides. Mutations in <i>acd-5</i> cause decreased lifespan and healthspan and a reduction in dauer entry in response to crude dauer pheromone. This suggested that the two genes were unlikely to be working in the same pathway, but do function in similar pathways. <br><br> Calcium imaging is a technique used in <i>C. elegans</i> to measure responses in excitable cells, in this case in neurons. Many calcium indicators are available for use in this technique, one in particular is GCaMP. GCaMP has undergone many rounds of targeted mutations with the aim to increase the molecule’s dynamic range and dissociation constant. At the time of commencing this project, new variants of GCaMP, known as GCaMP6s, became available, and had yet to be tested in <i>C. elegans</i> neurons. The effectiveness of a total of 6 new variants was tested in the gentle touch neurons of <i>C. elegans</i>. It was found that the alterations made to GCaMP5G in order to make the GCaMP6 variants did not result in improved dynamic range or dissociation constant in the PLM of <i>C. elegans</i>.