Evolution of lateralized gustation in nematodes

Abstract

Animals with small nervous systems have a limited number of sensory neurons that must encode information from a changing environment. This problem is particularly exacerbated in nematodes that populate a wide variety of distinct ecological niches but only have a few sensory neurons available to encode multiple modalities. How does sensory diversity prevail within this constraint in neuron number? To identify the genetic basis for patterning different nervous systems, we demonstrate that sensory neurons in Pristionchus pacificus respond to various salt sensory cues in a manner that is partially distinct from that of the distantly related nematode Caenorhabditis elegans. Previously we showed that P. pacificus likely lacked bilateral asymmetry (Hong et al., 2019). Here, we show that by visualizing neuronal activity patterns, contrary to previous expectations based on its genome sequence, the salt responses of P. pacificus are encoded in a left/right asymmetric manner in the bilateral ASE neuron pair. Our study illustrates patterns of evolutionary stability and change in the gustatory system of nematodes.

Keywords: Pristionchus pacificus; calcium imaging; developmental biology; lateral asymmetry.

Figure 1.. A comparison of chemotaxis responses to water-soluble ions between P. pacificus and C. elegans.

J4 to adult hermaphrodites from the two species responded to NH₄I, LiCl, and acetates significantly differently. Using two-way ANOVA, significant difference found between wildtype P. pacificus and C. elegans for the same salt is indicated above each pair (*p < 0.05, ****p < 0.0001), while the differences within P. pacificus is as follows: all salts showed difference when compared to NaAc and to NH₄Ac (****p < 0.0001), but not between NH₄Ac and NaAc. Both LiCl and NaCl attraction are significantly lower than NH₄Cl (*p < 0.05) and NH₄I (***p < 0.001). Error bars denote standard error of the mean and the sample sizes are indicated on the bottom.

Figure 2.. P. pacificus che-1 expression in ASE and AFD amphid neurons.

(A, B) The che-1::GFP marker in the che-1::RCaMP reporter strain is expressed in the ASE and AFD amphid neurons. (C) che-1::GFP expression is detectable in the morphologically distinct AFD neurons with ‘finger’-like dendritic endings. (D, E) Immunostaining of CHE-1::ALFA shows two pairs of amphid neuron cell bodies corresponding to the ASE and AFD neurons; dorsal–ventral view (n = 114). (F) The loss of che-1 results in loss che-1::RFP expression in the ASE (circle) while retaining reduced AFD expression. (G) ttx-1p::RFP expression in the AFD neurons with ‘finger’-like dendritic endings. Inset shows expanded inverted black–white image of the AFD dendritic ending. (H) AFD expression of the same ttx-1::RFP animal shown in (G) in a different plane with cell body in focus. (I, J) Immunostaining of TTX-1::ALFA (red) shows one pair of amphid neuron cell bodies co-localizing with the anterior pair of che-1::GFP-expressing AFD neurons (yellow); dorsal–ventral view (n = 13). Anterior is left and the scale bar in (C) represents 50 µm for all panels except for the G inset, which is 5 µm.

Figure 2—figure supplement 1.. Co-expression of gcy-22.3p::GFP and ttx-1p::RFP in ASER.

A representative F₁ male animal from a cross between the two reporter strains (n = 5). (A) gcy-22.3p::GFP. (B) ttx-1p::RFP. (C) Overlay of gcy-22.3p::GFP and ttx-1p::RFP. (D) Overlay of gcy-22.3p::GFP, ttx-1p::RFP, and DIC. Scale bar in (C) represents 50 µm for all panels.

Figure 3.. che-1 expressing amphid neurons are required for sensing water-soluble ions in P. pacificus.

(A) The che-1 locus with CRISPR/Cas9-induced mutations in Exon 1. (B) The che-1 mutants show defects in attraction toward NH₄Br, NH₄Cl, and LiCl. Sample sizes are indicated below for attractants and above for repellent. (C) The che-1p::HisCl1 animals lose attraction toward NH₄Br, NH₄Cl, and NH₄I in a histamine-dependent manner. Sample sizes are indicated at the base of each bar. (D) A schematic map of the P. pacificus AM7 (ASE) and AM12 (AFD) amphid neurons that express the che-1p::RCaMP used in calcium imaging. The ASE axons are the only amphid axons in P. pacificus to cross each other over the dorsal lateral midline contralaterally. **p < 0.01, *p < 0.05, two-way ANOVA with Dunnett’s post hoc comparison showing alleles with significant difference to wildtype P. pacificus (PS312). ****p < 0.0001, two-way ANOVA showing significant difference between water control and histamine treatment.

Figure 3—figure supplement 1.. The che-1p::HisCl1 transgene is necessary for histamine-dependent knockdown of salt attraction.

Animals containing the che-1p::HisCl1 transgene and the co-injection marker egl-20p::RFP were scored separately on each assay plate. Unpaired t-test between histamine and water treatment prior to chemotaxis assays (****p < 0.0001). Sample sizes for histamine RFP- (n = 23); RFP+ (n = 8) and water control RFP- (n = 31); RFP+ (n = 23).

Figure 3—figure supplement 2.. Molecular lesions for P. pacificus che-1 and gcy 22.3.

DNA alignment of the Ppa-che-1 and Ppa-gcy-22.3 alleles and their predicted amino acid changes.

Figure 4.. ASEL and ASER responses to different concentrations of NH₄Cl, NaCl, and NH₄I.

Average percent change in RCaMP fluorescence (dF/F) over time (seconds) of tracked ASE (left and right) sensory neurons in P. pacificus. Salts were presented at 10 s (‘ON’, left vertical line) for a duration of 20 s, and then removed (‘OFF’, right vertical line) for the remaining 30 s; the total recording time was 60 s. (A, B) ASEL and ASER neuron responses to high (250 mM, red) compared to low (25 mM, blue) concentrations of NH₄Cl. (C, D) ASEL and ASER neuron responses to NaCl. (E, F) ASEL and ASER neuron responses to NH₄I. Shaded ribbons represent 95% confidence intervals. Shaded ribbons in (E, F) have been cropped to maintain consistent y-axes across the plots, allowing for easier comparison. Sample sizes are indicated (n).

Figure 4—figure supplement 1.. Negative controls of ASE and AFD neuron responses to green light.

Average percent change in fluorescence (dF/F) over time (seconds) of tracked sensory neurons in P. pacificus (black line). Individual animals are represented by colored lines. No salt was presented; vertical lines indicate time points where salt was presented and removed for experimental samples, shown for comparison. csuEx93[Ppa-che-1p::optRCaMP] worms experienced the control solution for the duration of the recording and were imaged under green light. (A) ASE (n = 6). (B) AFD (n = 5).

Figure 4—figure supplement 2.. Individual traces and heatmaps of ASE responses to various salts.

Average percent change in fluorescence (dF/F) over time (seconds) of tracked ASEL and ASER neurons in P. pacificus (black line). Individual animals are represented by colored lines. For heatmaps, each row represents a single individual. Lighter colors (closer to 1) represent more positive dF/F values and darker colors (closer to 0) represent more negative dF/F values.

Figure 4—figure supplement 3.. Microfluidic apparatus used for calcium imaging.

(A) Schematic of the microfluidic apparatus to conduct calcium imaging while delivering stimuli directly to the nose of an immobilized worm. Syringes act as reservoirs for buffer solution (light blue), control solution (purple), and stimulant solution (red). Tubing inserts into the microfluidic PDMS chip. Panels (right) zoom in to depict a schematic of the microscope view of the PDMS chip: (top) fluid flow over the worm nose when stimulant is switched OFF (control solution flows over worm nose); (bottom) fluid flow over the worm nose when stimulant is switched ON (stimulant solution flows over worm nose). Created in BioRender. (B) Actual microscope view of PDMS chip design. The outer two channels hold buffer solution and can be switched open (ON) or closed (OFF) by the Valvebank. The inner two channels hold experimental solutions: the inner channel closer to the worm trap holds the control solution, and the inner channel farther from the worm trap holds the stimulant solution. (C) Actual microscope view of P. pacificus loaded into the PDMS chip while fluid is flowing. The PDMS chip features a U-shaped worm trap to facilitate loading the worm head-first, and a tapered opening to ensure the worm fits snugly and will not slide too far forward during recording.

Figure 5.. Combined AFD responses in comparison to ASE left or right neuron responses to NH₄Cl, NaCl, and NH₄I.

Average percent change in RCaMP fluorescence (dF/F) over time (seconds) as described in Figure 4. Averaged combined AFD (left and right neurons, orange) compared to left or right ASE (green) responses to (A, B) 250 mM NH₄Cl, (C, D) 25 mM NH₄Cl, (E, F) 250 mM NaCl, (G, H) 25 mM NaCl, (I, J) 250 mM NH₄I, and (K, L) 25 mM NH₄I. Shaded ribbons represent 95% confidence intervals. Shaded ribbons in (J, K) have been cropped to maintain consistent y-axes across the plots, allowing for easier comparison. Sample sizes are indicated (n).

Figure 5—figure supplement 1.. Individual traces and heatmaps of AFD responses to various salts.

Average percent change in fluorescence (dF/F) over time (seconds) of tracked left and right AFD neurons in P. pacificus (black line). Individual animals are represented by colored lines. For heatmaps, each row represents a single individual. Lighter colors (closer to 1) represent more positive dF/F values and darker colors (closer to 0) represent more negative dF/F values.

Figure 5—figure supplement 2.. AFDL/R responses to different concentrations of NH₄Cl, NaCl, and NH₄I.

Average percent change in RCaMP fluorescence (dF/F) over time (seconds) as described in Figure 4. Average AFDL and AFDR neuron responses to high (250 mM, red) and low (25 mM, blue) concentrations of salts: (A, B) NH₄Cl, (C, D) NaCl, and (E, F) NH₄I. Shaded ribbons represent 95% confidence intervals. Shaded ribbons in (D) have been cropped to maintain consistent y-axes across the plots, allowing for easier comparison. Sample sizes are indicated (n).

Figure 6.. The laterally asymmetric expression of gcy-22.3 is dependent on the zinc finger transcription factor CHE-1.

(A) The gcy-22.3::GFP marker is expressed exclusively in the right ASE neuron (ASER) (n > 200). (B) The gcy-22.3::GFP marker co-localizes with che-1::RFP expression in the ASER. (C) gcy-22.3::GFP expression is absent in the che-1(ot5012) mutant (n = 55). Anterior is left and dorsal is top. Scale bar in (A) represents 50 µm for all panels.

Figure 7.. ASE and AFD responses in wildtype compared to gcy-22.3 mutants.

Average percent change in RCaMP fluorescence (dF/F) over time (seconds) as described in Figure 4. (A) http://pristionchus.org/ Genome Browser view of the gcy-22.3 locus with the CRISPR/Cas9-induced mutations indicated with a red bar (Exon 5 of PPA04454 or Exon 4 of Contig12-snapTAU.506). (B, C) ASEL and ASER neuron responses to 250 mM NH₄Cl wildtype (gray) and gcy-22.3 mutants (magenta). (D, E) ASEL and ASER neuron responses to 250 mM NaCl in wildtype (gray) and gcy-22.3 mutants (magenta). AFD (combined left and right neurons) responses (F) to 250 mM NH₄Cl and (G) to 250 mM NaCl in wildtype (orange) and gcy-22.3 mutants (magenta). Shaded ribbons represent 95% confidence intervals. Sample sizes are indicated (n). For ASEL/R comparisons, bar plots represent the difference between the minimum % dF/F value 10 s pre-stimulus and maximum % dF/F value 10 s post-stimulus for either the (B) ON or (C, E) OFF stimulus. For AFD comparisons, bar plots represent maximum % dF/F values 10 s after the ON and OFF stimulus. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant difference. ‘ns’ indicate no significant difference. Comparisons between different genotypes were analyzed using unpaired t-test or Mann–Whitney test.

Figure 7—figure supplement 1.. AFDL and AFDR responses to NH₄Cl and NaCl in wildtype and gcy-22.3 mutant.

The combined AFD responses shown in Figure 7F, G have been separated into AFDL and AFDR. Average percent change in RCaMP fluorescence (dF/F) over time (seconds) as described in Figure 4. AFDL and AFDR neuron responses to (A, B) 250 mM NH₄Cl and (C, D) 250 mM NaCl in wildtype (orange) compared to gcy-22.3 mutants (magenta). Shaded ribbons represent 95% confidence intervals. Shaded ribbons in (D) have been cropped to maintain consistent y-axes across the plots. Sample sizes are indicated (n).

Figure 8.. Chemotaxis responses to individual ions in gcy-22.3 mutants.

Young adult hermaphrodite responses to salt gradients with or without various salts in background. *p < 0.05. Significant differences were found between wildtype PS312 and gcy-22.3 mutants to NH₄Cl by two-way ANOVA and Dunnett’s test. Each assay involves a minimum of 10 animals. Sample sizes for each condition are indicated on the bottom. Error bars denote standard error of the mean.

Author response image 1.