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doi: 10.1038/srep09564.

Dissection of C. elegans behavioral genetics in 3-D environments

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Dissection of C. elegans behavioral genetics in 3-D environments

Namseop Kwon et al. Sci Rep..

Abstract

The nematode Caenorhabditis elegans is a widely used model for genetic dissection of animal behaviors. Despite extensive technical advances in imaging methods, it remains challenging to visualize and quantify C. elegans behaviors in three-dimensional (3-D) natural environments. Here we developed an innovative 3-D imaging method that enables quantification of C. elegans behavior in 3-D environments. Furthermore, for the first time, we characterized 3-D-specific behavioral phenotypes of mutant worms that have defects in head movement or mechanosensation. This approach allowed us to reveal previously unknown functions of genes in behavioral regulation. We expect that our 3-D imaging method will facilitate new investigations into genetic basis of animal behaviors in natural 3-D environments.

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Figures

Figure 1
Figure 1. Worm Tracker 2.0.
3-D (a) Multi (triple)-view imaging system for 3-D worm tracker 2.0. A red arrowhead indicates the focal point. (b) Automatic 3-D tracking process of a single worm. (c) Reconstruction of a 3-D posture of a worm based on back-projections of x-, y- and z-views (scale bar represents 300 μm). (d) A trace and three representative reconstructed images of a worm moving in a gelatin cube (>2 cm3) for 3 minutes.
Figure 2
Figure 2. Locomotory behaviors of wild-type worms.
(a-c) Average speeds (a), reorientation frequencies (b) and curving rates for forward runs (c) of wild-type worms in 2-D and 3-D environments. Error bars represent SEM. n.s. (not significant), ***p < 0.0005, Mann-Whitney U-test. (d) Schematic for a best-fit ellipsoid of a 3-D postures of a worm. The orange, black and yellow axes represent three semi-principal axes with lengths R1, R2 and R3, respectively. Non-planar deviation (NPD) is defined as a value proportional to the ratio of R3 to R1. (e-f) Two representative “planar” (e) and “non-planar” (f) postures of worms with best-fit ellipsoids. The images were captured in two views: from the second and the first axes. (g) Relationship between NPDs and curving rates. The colors represent probability of the distribution. The curving rate has a positive correlation with normalized deviation as the Pearson correlation coefficient (0.28) indicates. The data were obtained by measuring the crawling locomotion of wild-type worms in 2-D and 3-D environments (n = 38 for 2-D and n = 35 for 3-D experiments).
Figure 3
Figure 3. Locomotory behaviors of mutants with abnormal head movements.
(a) Representative images of a wild-type, avab-10(e698) mutant, and aneat-4(n2474) mutant animals in 3-D environments (the scale bar represents 400 μm). Arrowheads indicate the heads of the worms and the inset is a magnified image of the bent head of avab-10 mutant (b) Curving rates for forward runs of wild-type and the mutant animals in 2-D and 3-D environments. (c) The average values of the non-planar deviation (NPD) of reconstructed 3-D postures. *p < 0.005, **p < 0.001, Mann-Whitney U-test. (d) Direction Autocorrelations (DAs), which represent directional changes during given time lags. (e) Differences between the DAs in 2-D and 3-D environments. Becausevab-10 mutants did not dig into the solidified 3% gelatin, wild-type,vab-10 mutant, andeat-4 mutant animals were loaded into 2% (w/v) gelatin solution at 20 °C, and habituated for 40 minutes for both 2-D and 3-D experiments in Fig. 3. We examined the following numbers of worms: n = 13 and 8 for wild-type animals; n = 8 and 14 forvab-10 mutants; n = 16 and 14 foreat-4 mutants for 2-D and 3-D experiments, respectively. Error bars represent SEM.
Figure 4
Figure 4. Locomotory behaviors of mechanosensary defective mutants.
(a-d) Curving rates for forward runs (a), non-planar deviations (NPDs) of 3-D posture (b), speeds (c), and reorientation frequencies (d) of indicated strains (n = 18 and 14 for wild-type; n = 17 and 14 formec-4(e1339); n = 15 and 11 formec-10(e1515); n = 20 and 24 forosm-9(ky10); n = 26 and 30 fortrpa-1(ok999); and n = 16 and 11 fortrp-4(sy695) mutants for 2-D and 3-D experiments, respectively). The reorientation frequencies were significantly reduced in 3-D compared to those in 2-D forosm-9 andtrp-4 mutants while showing little differences for wild type andmec-4,mec-10 andtrpa-1 mutants. In addition, we found thatocr-2(ak47) andglr-1(n2461) mutants, which are defective in OSM-9-mediated mechanosensation, also showed reduced reorientation frequencies in 3-D environments (Supplementary Fig. 6). (e) Reorientation frequencies of wild-type,trp-4(sy695) andtrp-4(sy695) mutant animals expressingtrp-4 in DVA neuron driven by atwk-16 promoter or in dopaminergic neurons driven by adat-1 promoter (n = 9 and 16 for wild-type; n = 26 and 21 fortrp-4 mutants; n = 25 and 28 fortrp-4(sy695); Ptwk-16(DVA)::trp-4; and n = 27 and 25 fortrp-4(sy695); Pdat-1::trp-4 for 2-D and 3-D experiments, respectively). Statistical analysis was performed to compare mutants with wild-type animals (a-b) or between 2-D and 3-D data (c-e) (*p < 0.005, **p < 0.001, ***p < 0.0005, Mann-Whitney U-test). Error bars represent SEM.
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