Data from: Dimensionality of locomotor behaviors in developing C. elegans
Abstract
Adult animals display robust locomotion, yet the timeline and mechanisms of how juvenile animals acquire coordinated movements and how these movements evolve during development are not well understood. Recent advances in quantitative behavioral analyses have paved the way for investigating complex natural behaviors like locomotion. In this study, we tracked the swimming and crawling behaviors of the nematode Caenorhabditis elegans from postembryonic development through to adulthood. Our principal component analyses revealed that adult C. elegans swimming is low dimensional, suggesting that a small number of distinct postures, or eigenworms, account for most of the variance in the body shapes that constitute swimming behavior. Additionally, we found that crawling behavior in adult C. elegans is similarly low dimensional, corroborating previous studies. Further, our analysis revealed that swimming and crawling are distinguishable within the eigenworm space. Remarkably, young L1 larvae are capable of producing the postural shapes for swimming and crawling seen in adults, despite frequent instances of uncoordinated body movements. In contrast, late L1 larvae exhibit robust coordination of locomotion, while many neurons crucial for adult locomotion are still under development. In conclusion, this study establishes a comprehensive quantitative behavioral framework for understanding the neural basis of locomotor development, including distinct gaits such as swimming and crawling in C. elegans.
https://doi.org/10.5061/dryad.stqjq2c8p
Description of the data and file structure
Data are organized into subfolders according to their locomotion type and developmental stage. Each .txt file contains tracking of a single worm with the first column showing timestamps and the next 10 columns showing the animal curvature in radians for that row. Files are named by date_strain_age_gait_file# or date_strain_age_gait_file#_worm# if there was more than one worm in a given assay. One adult swimming to crawling transition assay was included in this study for which there is one .txt file for.
Code/Software
Custom written Python scripts to reproduce figures will be made publicly available at the time of publication.
Preparation of Worms
Wild type hermaphrodite C. elegans (N2 Bristol) worms from the CGC (Minneapolis, MN, USA) were used for all assays. The worms were maintained at 15°C on 60mm NGM agarose plates with Escherichia coli OP50 lawns as food. To obtain worms at later stages of development, synchronization procedures were carried out in which 20 gravid hermaphrodites were placed on seeded NGM plates. After one hour, all worms were subsequently removed, leaving only eggs on the plates which were immediately put into a 20° incubator. After 21 hours of incubator growth time for late L1s, 29 hours for L2s, 42 hours for L3s, 50 hours for L4s, and 65 hours for adult worms, the synchronized animals were subjected to assays. Young L1 recently hatched animals were collected by transferring 50 gravid hermaphrodites to a seeded plate 24 hours before the experimental assay. On the day of the experiment, the plate with gravid hermaphrodites and laid eggs was washed with M9 buffer until all animals and bacteria were removed leaving only eggs on the plate. Over the course of an hour, plates were closely monitored for hatching and newly hatched young L1 animals were subjected to assays.
Assays
To obtain locomotion data across the stages of development, worms synchronized at each stage were subjected to swimming and crawling assays in which their movements were captured on video. All assays were conducted on NGM plates with no bacterial lawn. For crawling assays, worms were starved for one hour prior to the start of the assay. A platinum wire worm pick was used with Halocarbon 700 oil to transfer 20 worms onto a 90mm NGM plate in the absence of OP50. We took 1–2 minute-long video clips of each crawling worm. For swimming assays, each worm was transferred using a pick into a 5, 10, or 15µl drop of M9 buffer solution placed on the surface of the assay plate for L1-L2, L3, and L4-adult animals, respectively. M9 droplets were flattened with a worm pick in a circular motion beforehand to reduce glare. For each assay, one minute was allowed for the worm to acclimate to swimming conditions before 1–2 minutes of the worm's locomotion was tracked and analyzed. For gait transition assays, we followed the crawling protocol for adult animals, however, we added a 1.5µl drop of M9 buffer to the plate in the worm’s path following previously reported protocols.
Tracking
WormLab imaging stations were used in conjunction with WormLab software (both from MBF Bioscience) to capture videos of the worms and subsequently track the curvature data of each worm. Additionally, for young and late L1 larval assays, a macro lens (LAOWA 25mm F2.8 2.5-5x ULTRA MACRO) at 2.5x magnification was mounted instead of the default lens in order to capture the small worms at high resolution. All videos were taken at 1200x1600 resolution and 14 frames per second. To ensure worms remained in the field of view, assay plates were gently moved if necessary. For each worm, the angles between each of the eleven segments along the length of the animal (as defined by the WormLab software) were used to define the worm's curvature for each video frame. We segmented the animal into 5, 9, 11, 17, and 33 segments and found no significant differences in eigenworm shape analyses with more than 11 segments on a small held-out test data set. Subsequently, we proceeded with 11 segments (10 subsequent segment angles) for the remainder of the study.