A conserved neuropeptide system links head and body motor circuits to enable adaptive behavior
- Department of Neurobiology, University of Massachusetts Chan Medical School, United States
- Department of Biological and Physical Sciences, Assumption University, United States
- Department of Biology, University of Leuven (KU Leuven), Belgium
Figures
Figure 1 with 2 supplements
NLP-12/CCK induced locomotor responses require functional ckr-1 signaling.
(A) Representative movement trajectories of wild-type (black), nlp-12(OE) (red), nlp-12(OE);ckr-1(lf) (blue), nlp-12(OE);ckr-2(lf) (orange), and nlp-12(OE);ckr-1(lf);ckr-2(lf) (green) animals during …
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Figure 1—source data 1
Source data for body bending amplitude (Figure 1B).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig1-data1-v2.xlsx
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Figure 1—source data 2
Source data for frequency of bending angles (Figure 1D).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig1-data2-v2.xlsx
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Figure 1—source data 3
Source data for in vitro analysis of CKR-1 activation (Figure 1E).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig1-data3-v2.xlsx
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Figure 1—source data 4
Source data for in vitro analysis of CKR-2 activation (Figure 1F).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig1-data4-v2.xlsx
Figure 1—figure supplement 1
CKR-1 and CKR-2 GPCRs share similarity with vertebrate CCK GPCRs.
(A) Dendrogram (generated using Phylogeny,fr Dereeper et al., 2008). showing the predicted relationship between Drosophila (Dm_CCKLR-1/2), C. elegans (Ce_CKR-1/2), mouse (Mm), and human (Hs) CCK1/2R …
Figure 1—figure supplement 2
NLP-12 peptides activate CKR-1 and CKR-2 in vitro.
NLP-12–1 and NLP-12–2 elicit Ca2+ responses in cells expressing CKR-1 or CKR-2, but not in cells transfected with an empty pcDNA3.1 vector. Bar graphs indicate the ratio of total Ca2+ response of …
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Figure 1—figure supplement 2—source data 1
Source data for in vitro controls (ratio of total calcium response).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig1-figsupp2-data1-v2.xlsx
Figure 2
CKR-1 and CKR-2 differentially regulate head and body bending during basal locomotion.
Schematics showing body bending (A) and head bending (B) angles (solid orange circles indicate the vertices and measured angle in blue) quantified during single worm track analyses of movement (5 …
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Figure 2—source data 1
Source data for body bending measurements during single worm tracking of basal locomotion (Figure 2A).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig2-data1-v2.xlsx
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Figure 2—source data 2
Source data for head bending measurements during single worm tracking of basal locomotion (Figure 2B).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig2-data2-v2.xlsx
Figure 3 with 3 supplements
NLP-12/CCK food search responses are mediated through the GPCR CKR-1.
(A) Schematic of the food search assay indicating the time intervals when reorientations were scored. Wild-type animals increase reorientations during the first 5 min (0–5 min) after removal from …
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Figure 3—source data 1
Source data for reorientations quantified during area restricted search (0–5 min off food, Figure 3C).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig3-data1-v2.xlsx
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Figure 3—source data 2
Source data for reorientations quantified during dispersal (30–35 min off food, Figure 3E).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig3-data2-v2.xlsx
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Figure 3—source data 3
Source data for % change in reorientations from mean quantified for DVA photostimulation (Figure 3F).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig3-data3-v2.xlsx
Figure 3—figure supplement 1
Sequential snapshots of frames from a representative reorientation, for forward reorientations (A) and reversal-coupled omega turn mediated reorientations (B).
Frame #s and time points are indicated in each panel. Frame numbers and time points indicated are relative to the first image in each sequence, which represents the start point (frame 0, time 0 s) …
Figure 3—figure supplement 2
NLP-12 signaling through CKR-1 promotes forward reorientations.
(A) Quantification of reorientations during ARS (0–5 min following removal from food) compare to animals on food. Note that the increased number of forward and reversal coupled reorientations. Bars …
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Figure 3—figure supplement 2—source data 1
Source data for reorientations quantified on food and during area restricted search (0–5 min off food, Figure 3—figure supplement 2A).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig3-figsupp2-data1-v2.xlsx
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Figure 3—figure supplement 2—source data 2
Source data for reorientations quantified during area restricted search (0–5 min off food, Figure 3—figure supplement 2B).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig3-figsupp2-data2-v2.xlsx
Figure 3—figure supplement 3
NLP-12 released from DVA acts selectively through CKR-1 to promote reorientations.
(A) Quantification of reorientations during ARS (0–5 min following removal from food) for the genotypes indicated. Rescue refers to transgenic expression of wild-type ckr-1 or ckr-2 in ckr-1(ok2502);…
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Figure 3—figure supplement 3—source data 1
Source data for reorientations quantified during area restricted search (0–5 min off food, Figure 3—figure supplement 3A).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig3-figsupp3-data1-v2.xlsx
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Figure 3—figure supplement 3—source data 2
Source data for reorientations quantified during area restricted search (0–5 min off food, Figure 3—figure supplement 3B).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig3-figsupp3-data2-v2.xlsx
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Figure 3—figure supplement 3—source data 3
Source data for reorientations quantified during area restricted search (0–5 min off food, Figure 3—figure supplement 3C).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig3-figsupp3-data3-v2.xlsx
Figure 4
Elevated CKR-1 signaling enhances bending angle and amplitude in an nlp-12 dependent manner.
(A) Representative movement trajectories of wild-type (black), ckr-1(OE) (blue) and ckr-1(OE); nlp-12(lf) (green) animals for 30 s on NGM agar plates seeded with OP50 bacteria. ckr-1(OE) refers to …
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Figure 4—source data 1
Source data for frequency of bending angles (Figure 4B).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig4-data1-v2.xlsx
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Figure 4—source data 2
Source data for body bending amplitude (Figure 4C).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig4-data2-v2.xlsx
Figure 5 with 2 supplements
ckr-1 functions in the SMD head motor neurons to modulate body bending.
(A) Confocal maximum intensity projection of adult expressing the Pckr-1::ckr-1::SL2::GFP reporter. Note that the expression in multiple head neurons (white box) and a subset of ventral nerve cord …
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Figure 5—source data 1
Source data for body bending amplitude (Figure 5C).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Neuronal expression of CKR-1 and CKR-2.
(A) Confocal maximum intensity projections of a segment of the ventral nerve cord of a transgenic animal co-expressing Pckr-1::ckr-1::SL2::GFP and the cholinergic reporter Pacr-2::mCherry. ckr-1 is …
Figure 5—figure supplement 2
CKR-1 and CKR-2 expression are largely non-overlapping.
Confocal maximum intensity projections of transgenic worm expressing Pckr-1::ckr-1::SL2::mCherry and Pckr-2::GFP. (A) ckr-1 and ckr-2 expression in the entire worm. Both ckr-1 and ckr-2 are highly …
Figure 6 with 1 supplement
Ablation of SMD motor neurons abolishes the effects of ckr-1 overexpression.
(A) Representative tracks (1 min) for indicated genotypes. Asterisks indicate the position of animal at the beginning of recordings. Note that the increased reorientations and body bending depth in …
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Figure 6—source data 1
Source data for frequency of bending angles (Figure 6B).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig6-data1-v2.xlsx
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Figure 6—source data 2
Source data for frequency of bending angles (Figure 6E).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig6-data2-v2.xlsx
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Figure 6—source data 3
Source data for bending amplitude (Figure 6F).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig6-data3-v2.xlsx
Figure 6—figure supplement 1
Effects of ckr-1(OE) are dependent on NLP-12 and miniSOG expression alone does not alter SMD morphology or behavior.
(A) Representative tracks (30 s) for transgenic animals with high levels of cell-specific ckr-1 overexpression (Pflp-22∆4::ckr-1) in wild-type (top) or nlp-12 deletion background (bottom). Asterisks …
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Figure 6—figure supplement 1—source data 1
Source data for frequency of bending angles (Figure 6—figure supplement 1B).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig6-figsupp1-data1-v2.xlsx
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Figure 6—figure supplement 1—source data 2
Source data for frequency of bending angles (Figure 6—figure supplement 1C).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig6-figsupp1-data2-v2.xlsx
Figure 7 with 1 supplement
NLP-12/CKR-1 excitation of the SMD neurons promotes reorientations.
Total reorientations measured during 0–5 min following removal from food for the genotypes indicated. ckr-1 rescue refers to expression of wild-type ckr-1 (5 ng/µl) in ckr-1(ok2502) animals using …
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Figure 7—source data 1
Source data for reorientations quantified during area restricted search (0–5 min off food, Figure 7A).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig7-data1-v2.xlsx
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Figure 7—source data 2
Source data for reorientations quantified during SMD photostimulation (Figure 7C).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig7-data2-v2.xlsx
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Figure 7—source data 3
Source data for reorientations quantified during area restricted search upon SMD silencing (0–5 min off food, Figure 7D).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig7-data3-v2.xlsx
Figure 7—figure supplement 1
SMD activation modestly impacts body bending.
(A) Average body bending angle distribution (mean ± SEM) plotted for wild-type control animals (soid black circles, n=8) and Pflp-22∆4::ckr-1 (solid orange squares, n=8). Low level (5 ng/µl) …
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Figure 7—figure supplement 1—source data 1
Source data for frequency of bending angles (Figure 7—figure supplement 1A).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig7-figsupp1-data1-v2.xlsx
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Figure 7—figure supplement 1—source data 2
Source data for body bending amplitude quantified during SMD photostimulation (Figure 7—figure supplement 1B).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig7-figsupp1-data2-v2.xlsx
Figure 8 with 1 supplement
Elevated activity in SMD motor neurons during ARS promotes reorientations.
(A–C) Representative heat maps showing activity of SMD neurons in transgenic animals (Pflp-22∆4::GCaMP6s::SL2::mCherry) during ARS (A) and dispersal (B) for wild type, and ARS for ckr-1(ok2502) (C). …
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Figure 8—source data 1
Source data for GCaMP6s/mCherry ratio during SMD calcium imaging (Figure 8A–D).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig8-data1-v2.xlsx
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Figure 8—source data 2
Source data for mean GCaMP6s/mCherry ratio during SMD calcium imaging (Figure 8E).
- https://cdn.elifesciences.org/articles/71747/elife-71747-fig8-data2-v2.xlsx
Figure 8—figure supplement 1
Representative calcium signals (GCaMP6s/mCherry ratio) for wild-type ARS, wild-type dispersal, and ck-1(lf) ARS.
Corresponding behaviors are annotated by shading as indicated.
Figure 9
Proposed model for NLP-12 action through CKR-1 and CKR-2.
During basal locomotion, NLP-12 activation of CKR-1 and CKR2 GPCRs in ventral nerve cord motor neurons regulates body bending. During local searching, NLP-12 acts primarily through CKR-1 in SMD …
Videos
Video 1
Representative 20-s video showing locomotion on food of animal overexpressing nlp-12.
Video has been sped up 4×.
Video 2
Representative 20-s video showing locomotion of wild-type animal during area restricted search (0–5 min off food).
Video has been sped up 4×.
Video 3
Representative 20-s video showing locomotion of wild-type animal during dispersal (30–35 mi off food).
Video has been sped up 4×.
Video 4
Representative 20-s video showing locomotion on food of animal overexpressing ckr-1.
Video has been sped up 4×.
Video 5
Representative 20-s video showing locomotion on food of animal overexpressing ckr-1 in the SMD motor neurons.
Video has been sped up 4×.
Video 6
Representative 20-s video showing locomotion on food of animal in the absence (left) and during SMD photostimulation (right).
Video has been sped up 4×.
Video 7
Representative 20-s video showing simultaneous post hoc tracking of mCherry and GCaMP6s fluorescence for ratiometric calcium imaging analysis.
Video has been sped up 4×.
Video 8
Representative 20-s video showing tracking locomotion of animal overexpressing nlp-12 in WormLab to analyze body bending.
Video has been sped up 4×.
Video 9
Representative 20-s video showing single worm tracking of wild-type animal during basal locomotion on food to analyze body bending and head bending.
Video has been sped up 4×.
Additional files
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Supplementary file 1
Stains generated/used in this work.
- https://cdn.elifesciences.org/articles/71747/elife-71747-supp1-v2.docx
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Supplementary file 2
Identification (method of ID, marker and strain indicated for each neuron) to determine ckr-1 expressing neurons.
* Indicated strains were crossed into ufIs141 (Pckr-1::ckr-1::SL2::GFP) to generate strains to determine colocalization. #+ or – indicates presence or absence of ckr-1 expression in identified neuron. * Indicated strains were crossed into ufIs141 to generate strains to determine colocalization, #+ indicates ckr-1 expression, - indicates absence.
- https://cdn.elifesciences.org/articles/71747/elife-71747-supp2-v2.docx
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Supplementary file 3
Promoters used in ckr-1(OE) screen (Figure 5C) indicating expression pattern.
**Bold indicates neurons where ckr-1 is expressed.
- https://cdn.elifesciences.org/articles/71747/elife-71747-supp3-v2.docx
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Supplementary file 4
Plasmid constructs used in cell specific ckr-1(OE) screen or cell-specific rescue (Figures 5C and 7A).
For cell specific overexpression or rescue of ckr-1, ckr-1 minigene was expressed under indicated promoters. Entry vectors containing promoters recombined with destination vectors pRB12 or pRB13 for cell-specific overexpression or rescue of ckr-1.
- https://cdn.elifesciences.org/articles/71747/elife-71747-supp4-v2.docx
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Supplementary file 5
Promoter lengths and primer information for promoters used.
- https://cdn.elifesciences.org/articles/71747/elife-71747-supp5-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/71747/elife-71747-transrepform1-v2.pdf
