Article Figures & Data
Figures
- Figure 1.
Visual representation of the neuronal tracing algorithm and the confocal image of the C. elegans touch receptor neurons. A, Overview of C. elegans ALM and PLM touch receptor neurons. Only the left ALM(L) and PLM(L) neurons are shown in green. For detailed workflow for image acquisition and processing of ALM and PLM neuronal morphology see Extended Data Figure 1-1. B, Z-projection of a typical ALM neuron at day 8 of adulthood. C, Z-projection of a typical PLM neuron at day 8 of adulthood. D, E, Output of the APP2 neuron-tracing algorithm of region of interest shown in B, C, respectively (white rectangles), consists of a tree structure of connected nodes shown in red. F, H, Classified ALM neuronal tree with main-branch (gray), soma-nodes (white) and soma outgrowth (blue) in frontal (F) and top (H) view. Quantified morphologic features (soma-volume, soma outgrowth count and soma outgrowth length) are indicated in (F) and corresponding ground truth is shown in Extended Data Figure 1-2. G–I, Classified PLM tree with main-branch (gray), neurite-outgrowth (red) crossing PVM neuron (yellow) and sharp bends (blue) in lateral (G) and dorsal (I) view. Quantified morphologic features (bend count, neurite-outgrowth count and neurite-outgrowth length) are indicated in G and corresponding ground truth and quantification of sharp bends are shown in Extended Data Figures 1-2, 1-3. The heterogeneity of these age-related morphologic changes is illustrated by a collage of randomly selected z-projection of these neurons depicted in Extended Data Figure 1-4. Scale bars = 10 μm.Figure Contributions: Max Hess made all the figures.
- Figure 2.
The ALM neuron soma volume and the number and length of soma outgrowths are less effected by aging in long-lived C. elegans. A, Overview of quantified morphologic features in ALM neurons. Inset shows enlarged soma region with quantifications indicated (soma outgrowth count and length, blue; soma-volume, green). B, Soma outgrowth counts on days 1 and 8 of adulthood for WT and daf-2(e1370) mutants. Both conditions show a significant increase in soma outgrowth counts during aging (WT, p = 3.17e-15; daf-2(e1370), p = 1.94e-09). There is no significant difference between genotypes at day 1 (p = 0.376), but at day 8, soma outgrowth counts are significantly lower for daf-2(e1370) mutants compared to WT (p = 2.08e-03). For direct comparison of this data with previous studies, we replotted this data in percentage of ALM neurons with soma outgrowths (Extended Data Fig. 2-1). C, Lengths of individual soma outgrowths increase with age. This effect is statistically significant in WT animals (p = 2.20e-07), but no statistical significance is reached for daf-2(e1370) mutants (p = 0.149). At day 8 of adulthood, WT animals have significantly longer soma outgrowths (p = 3.38e-02). Error bars indicate bootstrapped 95% confidence interval of the mean, n is the number of outgrowth events scored. D, ALM soma volume decreases between day 1 and day 8 of adulthood (WT, p = 9.55e-12; daf-2(e1370), p = 1.52e-08), whereas WT somas are smaller than daf-2(e1370) somas (day 1, p = 3.60e-02; day 8, p = 2.44e-04). Error bars indicate bootstrapped 95% confidence interval of the mean, n is the number of neurons scored. E, F, Correlation of total soma outgrowth length (lengths of all soma outgrowths summed) versus soma volume of day 8 of adulthood C. elegans does not show a strong correlation for both WT (E, Pearson’s R = –0.127) and daf-2(e1370) mutants (F, Pearson’s R = –0.245). Data were collected in three independent trials and pooled for the analysis shown in this figure (B–F). ns = p > 0.05, * = p < 0.05, *** = p < 0.001, **** = p < 0.0001.Figure Contributions: Max Hess made all the figures.
- Figure 3.
Age-related morphologic abnormalities of PLM neurons are less severe in long-lived daf-2 mutants. A, Overview of morphologic features quantified in PLM neurons. Inset shows enlarged rectangular region with quantifications indicated (neurite-outgrowth count and length, red; sharp bends, blue; beads not shown). Yellow structure is the crossing PVM neuron. B, Neurite outgrowth counts at days 1 and 8 of adulthood for WT and daf-2(e1370) mutants. WT animals showed a significant increase in the number of neurite outgrowths between day 1 and day 8 (p = 3.15e-04), and higher neurite outgrowth counts than daf-2(e1370) mutants at day 8 (p = 1.12e-03). For direct comparison of this data with previous studies, we replotted this data in percentage of PLM neurons with neurite outgrowths (Extended Data Fig. 3-1). C, A significant decrease in bead density (bead count divided by the total length of the neurite) is found for WT animals (p = 8.25e-03), but not for daf-2(e1370) mutants comparing day 1 to day 8, respectively. However, no significant difference between genotypes at day 1 or at day 8 was found. Error bars are 95% bootstrapped confidence interval of the mean, n is the number of PLM neurons scored. D, Both WT and daf-2(e1370) mutants showed the same density of sharp bends (bend count divided by the total length of the neurite) at day 1 of adulthood (p = 0.903), whereas at day 8 of adulthood, there is a significant increase in bend density (day 1 vs 8: WT, p = 5.87e-18 vs daf-2, p = 8.34e-06). However, daf-2(e1370) mutants seems to protect against age-related wrinkly appearance of neuronal processes (daf-2 vs WT, p = 3.85e-07). This is in contrast to daf-2(RNAi) shown in Extended Data Figure 3-2. Error bars are 95% bootstrapped confidence interval of the mean, n is the number of PLM neurons scored. Data were collected in three independent trials and pooled for the analysis shown in this figure (B–D). See Extended Data Figure 3-3 for different angle threshold settings and Extended Data Figure 3-4 the histogram of angles and corresponding cumulative distributions. The waviness caused by osmotic shrinkage of C. elegans looks different to the age-related sharp bends (Extended Data Fig. 3-5). ns = p > 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Figure Contributions: Max Hess made all the figures.
- Figure 4.
Correlation of sharp bends with neurite outgrowths of PLM neurons during aging. A, Illustration depicting the procedure of determining the position of sharp bends (blue circles) and neurite outgrowths (red stars) along PLM neurons. The neuron was imagined to be stretch out along one dimension and the distances to the most distal point of the neuron was recorded. Furthermore, the distance to the nearest bend was recorded for every neurite outgrowth (green). B, C, Distribution of bends along the neurite for WT and daf-2(e1370) mutants at day 1 (B) and day 8 (C) shows distribution that bend occurrence is skewed toward the distal end of the neuron. C, At day 8 of adulthood, WT neurons (gray bars) not only showed a higher count of sharp bends compared to daf-2(e1370) mutants (red bars), but their distribution was shifted toward the proximal region of the neuron (Kolmogorov–Smirnov two-sample p = 3.20e-125). n indicates the number of sharp bend events evaluated. D, Distribution of outgrowth events along the neurite for WT and daf-2(e1370) mutants were not skewed toward the distal end. E, Plot of the 19 WT day-8 PLM neurons that showed neurite branching. Black lines represent the main branch stretched out along one dimension, neurite outgrowths (red stars) and sharp bends (blue circles) are plotted at their position along the neurite illustrates that outgrowth events often occur in close proximity to bends. F, CDFs of the distance to bends (confer A) and the ESD between bends. If outgrowths occurred independently of bends (meaning they were positioned randomly in the space between bends), one would expect the CDF to coincide with the empty space transformation. The course of the CDF compared to the ESD indicates that neurite outgrowths and sharp bends co-occur. As a statistical test, we assumed an interaction distance of 1 μm (inset, blue dotted line) and evaluated the individual CDFs and ESDs of the 19 WT day-8 neurons at that distance. A Wilcoxon signed-rank test showed that the percentage of outgrowth events that were 1 μm or closer to a sharp bend was significantly higher than one would expect if they were distributed randomly with respect to sharp bends (p = 2.90e-03). Data were collected in three independent trials and pooled for the analysis shown in this figure (B–F). Figure Contributions: Max Hess made all the figures.
Extended Data
Extended Data Figure 1-1
Workflow of the processing pipeline. A, Schematic representation of four out of six touch receptor neurons of C. elegans. ALM and PLM neurons that were used for image processing are shown in green. B, Three confocal image stacks were recorded per C. elegans (blue squares; A) leading to raw image stacks. C, Image stacks were pre-processed by stitching, gaussian blur, and manual removal of artefacts that could interfere with neuron tracing. D–F, A neuron tracing algorithm (APP2) was used to generate three-dimensional neuronal trees consisting of connected nodes (D), which were classified (E), and the morphology was quantified (E, F) using a semi-automated pipeline generated in this study. For a more detailed description, please see Materials and Methods. Download Figure 1-1, EPS file.
Extended Data Figure 1-2
Ground truth validation comparing automated results with manual and visually observed results. A, B, Pair-wise plot (A) and scatter-plot (B) of soma outgrowth lengths in automatic measurements and manually traced branches using the ImageJ “Simple neurite tracer” plug-in. C, D, Pair-wise plot (C) and scatter-plot (D) of soma volumes measured automatically and manually segmented soma. Rp is Pearson’s correlation coefficient. E, Bend count for 12 PLM neurons evaluated by seven people manually (black, thin), mean of manual counts (black, thick) and automatic counts (green). Algorithmic counts are close to the mean of manual counts. Download Figure 1-2, EPS file.
Extended Data Figure 1-3
Two approaches for the quantification of sharp bends. A, left panel, For every node (red) along the main branch, an angle (α) was calculated by linearly approximating upstream and downstream nodes in a window of 2 μm (red and green). Middle panel, Colors represent the angles calculated for every node as specified in the first panel. Blue corresponds to wider angles, orange to narrow angles. Right panel, Nodes with minimum angles were selected sequentially (red dots), double counting was avoided by non-maximum suppression. B, Visualization of the main branch backbone (red) and B-spline approximations of with differing amounts of smoothing (s). Blue colors correspond to parts of low curvature, orange to high-curvature. Scale bar = 2 μm. Download Figure 1-3, TIF file.
Extended Data Figure 1-4
Heterogeneity of age-related morphological changes of observed in touch receptor neurons. Randomly chosen z-projections of WT C. elegans touch neurons at ages day 1 and day 8. Download Figure 1-4, TIF file.
- Extended Data File 1.
Code and instruction for the neuronal quantification analysis. The zip file includes the python code files for batch processing, beads, classify, clean up, kink positions, soma volume, utility, and waviness. In addition, a swc example image, readme, requirements, and license text files are also included. Figure Contributions: Max Hess acquired confocal images. Max Hess, Alvaro Gomariz, Orcun Goksel, and Collin Ewald devised the image analysis pipeline, which was implemented by Max Hess and Alvaro Gomariz. Max Hess and Collin Ewald wrote the legends in consultation with the other authors. Download Extended Data F, ZIP file.
Extended Data Figure 2-1
Comparison of ALM morphological changes of three-week-old C. elegans maintained at lower temperature. A, Percentage of ALM neurons with soma outgrowths of WT and long-lived daf-2(e1370) at day 1 and day 8 of adulthood at 25°C. Same data as in Figure 2B plotted as percentage to make it comparable to previous studies. B, ALM soma volume of animals kept at 15°C until day 21 of adulthood showed the significant difference in soma-volume between WT and long-lived daf-2(e1370) mutants. C, WT and long-lived daf-2(e1370) mutants showed no significant difference in the number of soma outgrowths at day 21 of adulthood at 15°C. D, Contrary to our results at day 8 of adulthood at 25°C, the lengths of soma outgrowths are longer in daf-2(e1370) mutants compared to WT at 15°C at day 21 of adulthood. Download Figure 2-1, EPS file.
Extended Data Figure 3-1
Comparison of PLM morphological changes of three-week-old C. elegans maintained at lower temperature. A, Percentage of PLM neurons with neurite outgrowths of WT and long-lived daf-2(e1370) at day 1 and day 8 of adulthood at 25°C. Same data as in Figure 3B plotted as percentage to make it comparable to previous studies. B, Measurements of individual neurite outgrowths lengths observed in WT and daf-2 day-8 animals raised at 25°C does not show a significant difference due to the overall low number of observations of neurite branches (especially in daf-2 mutants). C, Neurite outgrowth count of WT and daf-2 animals raised at 25°C for 21 d does not show a significant difference between conditions (p = 0.837; WT n = 17, daf-2 n = 16). D, Bend density of WT and daf-2 animals raised at 25°C for 21 d does not show a significant difference between conditions (p = 0.911). Download Figure 3-1, TIF file.
Extended Data Figure 3-2
Reducing insulin/IGF-1 signaling via daf-2 knock-down does not protect against age-dependent neuronal morphological abnormalities. A, Both empty vector control and daf-2 RNAi knock-down showed a significant increase in soma outgrowth counts between day 4 and day 13 of adulthood (empty vector control, p = 9.48e-03; daf-2 RNAi, p = 1.29e-03) but daf-2 RNAi knock-down did not have a significant effect (day 4, p = 0.684; day 13, p = 0.667). B, Only daf-2 RNAi knock-down showed a significant increase in neurite-outgrowth counts between day 4 and day 13 of adulthood (empty vector control, p = 7.56e-02; daf-2 RNAi, p = 1.71e-03), and showed higher outgrowth counts at day 13 (p = 2.78e-02). C, Both empty vector control and daf-2 RNAi knock-down showed a significant increase in bend counts between day 4 and day 13 of adulthood (empty vector control, p = 2.37e-08; daf-2 RNAi, p = 1.44e-08) but daf-2 RNAi knock-down did not have a significant effect (day 4, p = 0.102; day 13, p = 0.089). Download Figure 3-2, EPS file.
Extended Data Figure 3-3
Robustness of PLM bend densities across different angles threshold settings. Using the data from Figure 3D with different angle thresholds, ranging from 135° to 165°, are quantified and show similar results of bend density across genotype and age. As expected, increasing the threshold leads to higher bend densities, but the differences between conditions are insensitive to changes in threshold angle settings. Dots show individual measurements and blue error bars are bootstrapped 95% confidence interval. Please note, the differently scaled axes between the rows. Download Figure 3-3, TIF file.
Extended Data Figure 3-4
Histogram and CDFs across angles. Using data from Figure 3D, histogram and cumulative distribution of angles are plotted. A, Histogram showing angle measurements of all PLM main branch nodes for WT and daf-2(e1370) mutants at days 1 and 8 of adulthood. B, Corresponding cumulative distributions of all angles for WT and daf-2(e1370) mutants at days 1 and 8 of adulthood. Download Figure 3-4, TIF file.
Extended Data Figure 3-5
Osmotic shrinkage of C. elegans results in bending of PLM neurons that look different than the age-related sharp bends. A, Fluorescent image of a typical PLM neuron at day 4 of adulthood shows beads along the process but no sharp bends. B, Fluorescent image of a typical PLM neuron at day 13 of adulthood shows a couple of sharp bends along the process. C, Fluorescent image of a typical PLM neuron at day 3 of adulthood that was imaged upon placing the C. elegans in a drop of 1.7 M NaCl to induce osmotic pressure. The induced wrinkles appear different (rounder curvature as observed when neurons supercoil; Krieg et al., 2017; see Discussion) than the age-related morphology. For A–C, insets are 3× zoom of dashed rectangles. Scale bars = 20 μm. Download Figure 3-5, TIF file.
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