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Sexually dimorphic control of gene expression in sensory neurons regulates decision-making behavior in C. elegans

  1. Zoë A Hilbert
  2. Dennis H Kim Is a corresponding author
  1. Massachusetts Institute of Technology, United States
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Cite as: eLife 2017;6:e21166 doi: 10.7554/eLife.21166

Figures

Figure 1 with 1 supplement
Sex-specific expression of daf-7 in the ASJ neurons of adult males.

(A) pdaf-7::gfp expression pattern in hermaphrodites (left) and males (right). Filled triangles indicate the ASI neurons; dashed triangles indicate the ASJ neurons. Scale bar indicates 50 µm. (B) Maximum fluorescence values of pdaf-7::gfp in the ASI (left) and ASJ (right) neurons of age-matched adult hermaphrodites (grey) and males (black). *p<0.05 as determined by an unpaired t-test with Welch’s correction. Error bars indicate standard deviation (SD). n.s., not significant. (C) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of males through larval development and early adulthood. The developmental timeline of C. elegans is shown to scale on top. Time values indicate hours after an egg is laid. Error bars indicate SD. (D) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of both males and hermaphrodites on E. coli (yellow) or P.aeruginosa (green). ***p<0.001 as determined by unpaired t-tests with Welch’s correction. Error bars indicate SD.

https://doi.org/10.7554/eLife.21166.003
Figure 1—figure supplement 1
daf-7 expression in the ASJ neuron pair is specific to adult males.

(A–B) FISH images of endogenous daf-7 mRNA in adult hermaphrodite (A) and adult male (B). White dotted lines indicate outline of the ASJ neuron(s). Both images are a single slice through the animal where ptrx-1::gfp was visible and in focus in one or both ASJ neurons. Other neurons visible anterior to the ASI neurons are likely the OLQ neurons, and the cells posterior to the ASJ neurons that express daf-7 mRNA are likely the ADE neurons. Scale bar represents 25 µm. Orientation provided in (A) applies to both images. (C) Fluorescence quantification for daf-7-Cy5 probe in the ASJ neurons (as localized by ptrx-1::gfp) in males and hermaphrodites. ***p<0.001 as determined by unpaired t-test with Welch’s correction. Error bars represent SD. (D) pdaf-7::gfp expression pattern in an L4 larval male. Filled triangles indicate the ASI neurons. Scale bar (shown in C) indicates 50 µm. (E–F) Maximum fluorescence values of pdaf-7::gfp in the ASI (E) and ASJ (F) neurons of developing males. **p<0.01, ***p<0.001, as determined by unpaired t-test with Welch’s correction. Error bars represent SD. n.s., not significant.

https://doi.org/10.7554/eLife.21166.004
Figure 2 with 1 supplement
Genetic sex regulates male-specific expression of daf-7 in the ASJ neuron pair.

(A–D) Representative images of pdaf-7::gfp expression in: wild-type (WT) hermaphrodites (A) and males (B), nervous-system-masculinized hermaphrodites (C) and nervous-system-feminized males (D). Closed triangles indicate the ASI neurons; dashed triangles indicate the ASJ neurons. Scale bar indicates 50 µm. (E) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of control (black) and partially masculinized hermaphrodites (purple). Masculinization was effected by driving expression of fem-3 cDNA under pan-neural (left) and ASJ-specific (right) promoters. ***p<0.001 as determined by unpaired t-test with Welch’s correction. n.s., not significant. Error bars represent SD. (F) Maximum fluorescence values of pdaf-7::gfp in ASJ of control (black) and partially feminized males (orange). Feminization was effected by driving expression of tra-2IC under pan-neural (left), ciliated neuron (middle), and ASJ-specific (right) promoters. ***p<0.001 as determined by unpaired t-test with Welch’s correction. Error bars indicate SD.

https://doi.org/10.7554/eLife.21166.005
Figure 2—figure supplement 1
daf-7 expression in the ASJ neuron pair is regulated by TRA-1 but not by male-specific neurons.

(A) Representative image of pdaf-7::gfp expression pattern in tra-1(e1099) pseudomale. Filled triangles indicate the ASI neurons; dashed triangles indicate the ASJ neurons. Scale bar represents 50 µm. (B) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of WT, ceh-30(n4289), mab-3(e1240), and pkd-2(sy606);lov-1(sy582) mutant animals. Significance determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. Error bars represent SD. n.s., not significant.

https://doi.org/10.7554/eLife.21166.006
Figure 3 with 2 supplements
DAF-7/TGF-β is required for male mate-searching behavior.

(A) Schematic of mate-searching assay (top). Animals are placed individually into the center of a lawn of bacteria. The tracks of the animal are followed over time and scored for movement beyond 3 cm away from the food source. A representative data curve is shown on the bottom depicting hermaphrodites (solid black), males (dashed black), and daf-7(e1372) mutant males (blue). (B) Probability of leaving values for WT, daf-7 mutant, and daf-7;daf-3 double mutant males. Values plotted are the average + SEM for two independent experiments, n = 40 animals for all strains except daf-7(ok3125) where n = 29. *p<0.05, **p<0.01 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. n.s., not significant. (C) Probability of leaving values for two independent lines of male animals with genetic ablation of the ASJ neurons, compared with corresponding values for WT and daf-7 mutant males. Values plotted are the average + SEM for two independent experiments for daf-7 mutant animals and three independent experiments for WT control and ASJ ablation strains. n = 60 animals for all strains except daf-7(ok3125) where n = 29 animals. **p<0.01 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. One replicate was performed with the daf-7;daf-3 double mutant strains in 1B and controls (WT and daf-7) are identical, so probability values for that experiment were used in the averages shown in both B and C of this figure. (D) Probability of leaving values for daf-7(ok3125) mutant animals carrying transgenes expressing daf-7 cDNA specifically under the control of the indicated promoters. Values on graph are the average + SEM of two independent experiments with an n = 40 total animals for each strain. **p<0.01 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. n.s., not significant.

https://doi.org/10.7554/eLife.21166.007
Figure 3—figure supplement 1
daf-7 regulates mate-searching behavior in males.

(A) Representative mate-searching data for strains with genetic ablation of the ASJ neurons (shown in shades of orange). WT hermaphrodites (solid black), WT males (dashed black), and daf-7 mutant (blue) animals are included in each assay as controls. (B) Representative data for daf-7;daf-3 double mutant (green) in the mate-searching assay. (C) Representative data for daf-7 rescue strains (shown in shades of red). (D) Representative mate-searching curves for daf-1 mutants (purple). (E) Probability of leaving values for WT, daf-7, and daf-1 mutant animals. Values shown are the average + SEM of two independent experiments. n = 40 for WT and daf-7 mutants, n = 32 for daf-1(e1287) and n = 36 for daf-1(m402). *p<0.05 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test.

https://doi.org/10.7554/eLife.21166.008
Figure 3—figure supplement 2
daf-7 mutant animals and strains carrying genetic ablation of the ASJ neurons exhibit no locomotion defect.

(A) Schematic of locomotion assay. E. coli food (shown in yellow) was spread to the edges of the plate and a standard mate searching assay performed to determine animal movement on a full lawn of bacteria. (B–C) Raw data curves for daf-7 (blue) and ASJ-ablation (orange) males in locomotion assay. WT males shown in black for reference.

https://doi.org/10.7554/eLife.21166.009
Figure 4 with 1 supplement
Nutritional state regulates daf-7 expression in the ASJ neurons of males.

(A) Schematic of experiment design indicating timing and duration of starvation periods (left). FISH images of endogenous daf-7 mRNA in the ASJ neurons for each experimental condition (right). White dotted lines indicate outline of ASJ cell body as localized by ptrx-1::gfp. Scale bar represents 5 µm. (B) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons during re-feeding experiment. Re-fed animals were starved for a period of 24 hr before being reintroduced to E. coli for the indicated amount of time. ***p<0.001 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. Error bars indicate SD. n.s., not significant. (C) Representative curves of mate-searching data in fed and post-starvation WT males. Animals were starved for a period of 24 hr as in previous experiments before being assayed for mate-searching behavior.

https://doi.org/10.7554/eLife.21166.010
Figure 4—figure supplement 1
daf-7 expression is down-regulated by starvation specifically in the ASJ neurons.

(A) Representative FISH image of endogenous daf-7 mRNA in a starved adult male. White dotted line indicates outline of an ASJ neuron. Closed triangle indicates an ASI neuron. Other neurons visible anterior to the ASI neuron are likely the OLQ neurons, and posterior to the ASJ neuron are likely the ADE neurons. Scale bar represents 25 µm. (B) Fluorescence quantification for daf-7-Cy5 probe in the ASJ neurons (as localized by ptrx-1::gfp) of males fed and starved at various different points in their lives. ***p<0.001 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. Error bars represent SD. n.s., not significant. (C) Fluorescence quantification for daf-7-Cy5 probe in the ASI neurons of starved and fed males. Significance determined by unpaired t-test with Welch’s correction. Error bars represent SD. n.s., not significant.

https://doi.org/10.7554/eLife.21166.011
Figure 5 with 1 supplement
Prioritization of multiple inputs through the regulation of daf-7 expression in the ASJ neurons of males.

(A) Schematic of refeeding experimental design indicating timing and duration of each starving and feeding period (left). On right, representative images of daf-7 expression following 4 hr of post-starvation refeeding on E.coli (left) or P.aeruginosa (right). Closed triangles indicate the ASI neurons; dashed triangles indicate the ASJ neurons. Scale bar represents 50 µm. (B) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of fed and starved animals and animals that were starved for a period of 24 hr and then reintroduced either to the normal E. coli food source or to pathogenic P. aeruginosa. Images were taken for quantification after 4 hr of re-feeding. ***p<0.001 as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. Error bars indicate SD. n.s., not significant. (C) Representative curve of food-leaving data in starved animals on E. coli (yellow) and P. aeruginosa (green). Animals were starved as in previous experiments before being run in the assay.

https://doi.org/10.7554/eLife.21166.012
Figure 5—figure supplement 1
P. aeruginosa influences exploratory behavior and daf-7 expression in both males and hermaphrodites.

(A) Representative leaving curves for males (dashed) and hermaphrodites (solid) on E. coli (shades of grey) and P. aeruginosa (shades of green). All strains were fed in this experiment. (B) Probability of leaving values for males and hermaphrodites on E. coli and P. aeruginosa. All strains were fed in these experiments. Values plotted are the mean + SEM for two independent experiments, n = 40 animals for all conditions. *p<0.05 as determined by unpaired t-test with Welch’s correction. n.s., not significant. (C) Representative raw data curves for starved hermaphrodites on E. coli and P. aeruginosa. Fed hermaphrodites shown as control. (D) Maximum fluorescence of pdaf-7::gfp in the ASJ neurons of starved hermaphrodites re-fed E. coli or P. aeruginosa for four hours (compare to Figure 5B). Error bars indicate SD. (E) Maximum fluorescence values of pdaf-7::gfp in the ASJ neurons of fed and starved animals and animals that were starved for a period of 24 hr and then reintroduced to P. aeruginosa for the indicated amount of time. The experiment in Figure 4B was run in parallel, controls (fed and starved) were shared between conditions. These controls are shown in both figures for comparison purposes. *p<0.05 and ***p<0.001, as determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. Error bars indicate S.D.

https://doi.org/10.7554/eLife.21166.013
A hierarchy of inputs regulates daf-7 expression in the ASJ neurons to modulate exploratory behaviors.

We show that the information from many different sources both internal and external is integrated hierarchically in the regulation of daf-7 expression in the ASJ neuron pair. This hierarchical regulation of daf-7 in turn leads to a distinct prioritization of exploratory behaviors in the male worm.

https://doi.org/10.7554/eLife.21166.014

Additional files

Supplementary file 1

C. elegans strains used in this study

A comprehensive list of the strains used in this study. With the exception of CB1490, all strains are previously unpublished and were constructed for this study.

https://doi.org/10.7554/eLife.21166.015
Supplementary file 2

Oligos used for transgenic strain generation.

A comprehensive list of the oligos used for constructing the transgenic strains created for this study. All sequences are listed in the 5’ to 3’ direction.

https://doi.org/10.7554/eLife.21166.016

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