An hourglass circuit motif transforms a motor program via subcellularly localized muscle calcium signaling and contraction
- Howard Hughes Medical Institute, Department of Biology, McGovern Institute for Brain Research, Massachusetts Institute of Technology, United States
- Miller Institute, Helen Wills Neuroscience Institute, Department of Molecular and Cellular Biology, University of California, Berkeley, United States
- Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, United States
Figures
Figure 1 with 1 supplement
Worms spit as a consequence of the sustained contraction of anterior pharyngeal muscles.
(A) Anatomy of the C. elegans pharynx; a, anterior; d, dorsal. (B) Grinder pumping response to 365 nm UV light. Left, raster plot of individual animals, each grinder contraction shown by a tick; …
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Figure 1—source data 1
Source data for Figure 1.
- https://cdn.elifesciences.org/articles/59341/elife-59341-fig1-data1-v2.zip
Figure 1—figure supplement 1
The odor of hydrogen peroxide induces spitting.
(A) The odor of 9M hydrogen peroxide induces spitting. Animals are unc-29(e1072) mutants to facilitate video capture. n = four animals. (B) Raster plot showing occurrence of ‘closed’ (black) and …
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Figure 1—figure supplement 1—source data 1
Source data for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/59341/elife-59341-fig1-figsupp1-data1-v2.zip
Figure 2 with 2 supplements
Subcellularly localized muscle contractions and calcium increases open the pharyngeal valve to produce spitting.
(A) Anatomy of the anterior pharynx showing pharyngeal muscles pm1, pm2, and pm3. Double-headed arrows indicate orientation of muscle fibers. (B–E) Mock-ablated (n = nine animals), pm1-ablated (n = …
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Figure 2—source data 1
Source data for Figure 2.
- https://cdn.elifesciences.org/articles/59341/elife-59341-fig2-data1-v2.zip
Figure 2—figure supplement 1
Additional characterization of the effects of pm1 ablation and of the immobilized muscle calcium imaging assay.
(A–B) pm1-ablated animals lack the metastomal filter and thus ingest more material into the procorpus than mock-ablated controls do. Images were obtained from the first frame of the videos used to …
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Figure 2—figure supplement 1—source data 1
Source data for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/59341/elife-59341-fig2-figsupp1-data1-v2.zip
Figure 2—figure supplement 2
Characterization of the pharyngeal expression pattern of transgenes used in calcium imaging.
(A) Transgene nIs686 (gpa-16p::gcamp3) drives GCaMP expression in pharyngeal muscles pm2 and pm3 and in the mc1 marginal cells (not shown). Occasional dim expression was observed in pm1 (not shown). …
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Figure 2—figure supplement 2—source data 1
Source data for Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/59341/elife-59341-fig2-figsupp2-data1-v2.zip
Figure 3 with 1 supplement
Pharyngeally expressed gustatory receptor orthologs lite-1 and gur-3 play major and minor roles, respectively, in the light-induced spitting reflex.
(A–D) Light-induced spitting is reduced in gur-3 and lite-1 single mutants and eliminated in lite-1 gur-3 double mutants. n = 10 animals except ngur-3 = 9. (E–F) Expression of wild-type lite-1 (trans…
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Figure 3—source data 1
Source data for Figure 3.
- https://cdn.elifesciences.org/articles/59341/elife-59341-fig3-data1-v2.zip
Figure 3—figure supplement 1
Additional characterization of gustatory receptor ortholog function and expression.
(A) egl-47/gur-1(n1082dm) mutants were normal in burst pumping. n = 20 animals. (B) egl-47/gur-1(ok677) mutants were normal in burst pumping. n = 20 animals. (C) gur-3 is not required for …
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Figure 3—figure supplement 1—source data 1
Source data for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/59341/elife-59341-fig3-figsupp1-data1-v2.zip
Figure 4 with 2 supplements
The pharyngeal neuron M1 controls the spitting reflex.
(A) The anatomy of M1 (red) suggests that it controls spitting directly, as it is the only neuron to make neuromuscular junctions (bolded blue segment) with the region of pharyngeal muscle that …
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Figure 4—source data 1
Source data for Figure 4.
- https://cdn.elifesciences.org/articles/59341/elife-59341-fig4-data1-v2.zip
Figure 4—figure supplement 1
Additional characterization of the function of the M1 pharyngeal neuron in spitting and burst pumping.
(A) Overexpression of the mammalian caspase ICE in M1 via the transgene nEx2905 (M1promoter – lury-1::ice::sl2::mCherry) kills M1 with high efficiency and the M2 pharyngeal neurons with moderate …
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/59341/elife-59341-fig4-figsupp1-data1-v2.zip
Figure 4—figure supplement 2
Burst pumping responses of acetylcholine receptor mutants to UV light.
(A) lev-1/acr-1(e211) nicotinic acetylcholine receptor mutants were normal in burst pumping. n = 20 animals. (B) acr-2(n2595 n2420) nicotinic acetylcholine receptor mutants were normal in burst …
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Figure 4—figure supplement 2—source data 1
Source data for Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/59341/elife-59341-fig4-figsupp2-data1-v2.zip
Figure 5
The gur-3-Expressing I2 Neurons Can Activate M1 to Produce an Attenuated Form of Spitting.
(A–B) I2-specific expression of gur-3 restores light-induced spitting to lite-1 gur-3 mutants. n = five animals. (C) Quantification of the frequencies at which the metastomal filter was open in …
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Figure 5—source data 1
Source data for Figure 5.
- https://cdn.elifesciences.org/articles/59341/elife-59341-fig5-data1-v2.zip
Figure 6 with 1 supplement
The I4 neuron functions with the I2 neurons to promote spitting and inhibit feeding.
(A) Genetic ablation of the I2 neurons in a wild-type background does not eliminate light-induced spitting. n = five animals. (B) Genetic ablation of the I2 neurons of lite-1(ce314) mutants does not …
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Figure 6—source data 1
Source data for Figure 6.
- https://cdn.elifesciences.org/articles/59341/elife-59341-fig6-data1-v2.zip
Figure 6—figure supplement 1
Additional characterization of the effects of ablating the I2 and/or I4 neurons inlite-1mutant animals.
(A) M1 somatic GCaMP responses to light of individual mock-, I2-, I4-, and I2/I4-ablated animals. Each row represents a different animal; ΔF/F0 over time is indicated according to the heatmap at …
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Figure 6—figure supplement 1—source data 1
Source data for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/59341/elife-59341-fig6-figsupp1-data1-v2.zip
Figure 7
Models of spitting behavior and neural circuitry.
(A) Model for feeding and spitting. During feeding, the M1 neuron is inactive. Thus, the metastomal filter is partially closed, restricting particle influx, and the pharyngeal valve closes at the …
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Figure 7—source data 1
Source data for Figure 7.
- https://cdn.elifesciences.org/articles/59341/elife-59341-fig7-data1-v2.zip
Videos
Video 1
Feeding pumps in polystyrene bead feeding assay.
High frame rate video of C. elegans feeding in polystyrene bead feeding assay. The small, dark particles are polystyrene beads. During typical feeding pumps, beads accumulate in the buccal cavity. …
Video 2
Spitting pumps in polystyrene bead feeding assay.
High frame rate video of C. elegans spitting in response to light in polystyrene bead feeding assay. During spitting pumps, beads no longer accumulate in the buccal cavity, but instead freely enter …
Video 3
Feeding pumps in muscle motion visualization assay.
Video of C. elegans feeding beneath a coverslip. The pharyngeal lumen is visible as a white space when it is opened by the contraction of pharyngeal muscle. In feeding, the musculature of the …
Video 4
Spitting pumps in muscle motion visualization assay.
Video of C. elegans beneath a coverslip spitting in response to light. In contrast to feeding pumps, spitting pumps are characterized by the sustained contraction of the anterior musculature of the …
Video 5
‘Gulping’ pumps in polystyrene bead feeding assay.
High frame rate video of C. elegans ‘gulping’ in polystyrene bead feeding assay.
As in spitting pumps, beads pass freely through the buccal cavity and into the pharyngeal lumen. As in feeding pumps, this material is subsequently trapped and retained by the pharyngeal valve, resulting in ingestion. Video was recorded at 1000 frames per second. Playback is at 3% of original speed.
Video 6
pm2-ablated animals are defective in opening the metastomal filter during spitting behavior in polystyrene bead feeding assay.
High frame rate video of pm2-ablated C. elegans spitting in response to light in polystyrene bead feeding assay. In contrast to typical spitting pumps, beads accumulate in the buccal cavity, …
Video 7
A subset of pm1-ablated animals spit less efficiently.
High frame rate video of pm1-ablated C. elegans spitting in response to light in polystyrene bead feeding assay. At the end of the pump, ingested material is expelled, indicating that the pharyngeal …
Video 8
A subset of pm1-ablated animals exhibit normal spitting behavior.
High frame rate video of pm1-ablated C. elegans spitting in response to light in polystyrene bead feeding assay. Spitting behavior is comparable to that of mock-ablated animals. Animals carried …
Video 9
pm1/pm2 double-ablated animals can hold open the pharyngeal valve and spit.
High frame rate video of pm1/pm2 double-ablated C. elegans spitting in response to light in polystyrene bead feeding assay. At the end of the pump, the material ingested during the pump is spat out, …
Video 10
Light induces immobilized animals to spit.
Video of immobilized C. elegans spitting in response to 2 Hz flickered 365/485 nm light (i.e. the same as delivered to animals during calcium imaging). Animal was selected based on its ingestion of …
Video 11
Pharyngeal muscle calcium response to light (gpa-16p::gcamp3 transgene).
The pharyngeal muscle calcium response to light, as reported by fluorescence of GCaMP3 transgene nIs686 (gpa-16p::gcamp3), which is expressed in the pm2 and pm3 pharyngeal muscles, the mc1 marginal …
Video 12
Sustained, anteriorly localized calcium signals do not occur when animals are induced to pump using serotonin.
Animals carrying the muscle-GCaMP transgene nIs686 [gpa-16p::gcamp3; lin-15(+)] were immobilized in the presence of 10 mM serotonin and filmed at the onset of pumping. Video was recorded at four …
Video 13
Pharyngeal muscle calcium response to light (myo-2p::gcamp3 transgene).
The pharyngeal muscle calcium response to light as reported by fluorescence of GCaMP3 transgene cuIs36 (myo-2p::gcamp3), which is expressed specifically in the pm3s but not the pm2s and pm1. …
Video 14
Pharyngeal muscle calcium response to light (C32F10.8p::gcamp3 transgene).
The pharyngeal muscle calcium response to light, as reported by fluorescence of GCaMP3 transgene nEx3045 [C32F10.8p::gcamp3; lin-15(+)], which is expressed specifically in the pm3 pharyngeal muscles …
Video 15
The pharyngeal neuron M1 is activated by light (glr-2p::gcamp6s transgene).
The pharyngeal neuron M1 is acutely activated by light, as reported by fluorescence of GCaMP6s calcium imaging transgene nIs678 [glr-2p::gcamp6s; lin-15(+)]. Video was recorded at two frames per …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Caenorhabditis elegans) | N2 (wild type) | Horvitz lab collection | WBStrain00000001 | Laboratory reference strain Figure 1B–G; Figure 1—figure supplement 1A; Figure 2—figure supplement 1C and D; Figures 3A, G and H; Videos 1, 2 and 5 |
| Strain, strain background (C. elegans) | CB1072 | Horvitz lab collection | WBStrain00004240 | unc-29(e1072) Figure 1—figure supplement 1B and C; Videos 3 and 4 |
| Strain, strain background (C. elegans) | MT26279 | this study | n/a | lin-15(n765ts); nIs507 [inx-4promoter::gfp; lin-15(+)] 8x outcrossed to MT8189 Figure 2B–E; Figure 2—figure supplement 1A and B; Videos 6, 7 and 8, and 9 |
| Strain, strain background (C. elegans) | MT8189 | Horvitz lab collection | WBStrain00027314 | lin-15(n765ts) Used to outcross MT26279, MT23338, and MT24110 |
| Strain, strain background (C. elegans) | MT23338 | this study | n/a | nIs686 [gpa-16promoter::gcamp3::unc-54 3’ UTR; lin-15(+)] III; lin-15AB(n765ts) 5x outcrossed to MT8189 Figure 2H–J; Figure 2—figure supplement 1C, D and H; Figure 3I–N; Figure 4F and G, Videos 10 and 11 |
| Strain, strain background (C. elegans) | MT26375 | this study | n/a | lin-15AB(n765ts); nEx3045 [C32F10.8p::GCaMP3; lin-15(+)] Figure 2—figure supplement 1C, D and G; Figure 2—figure supplement 1D; Figure 2M and N; Video 14 |
| Strain, strain background (C. elegans) | MT25732 | this study | n/a | lin-15(n765ts); nIs864; nEx2905 [lury-1promoter::ice::sl2::mcherry::unc-54 3’ UTR; ttx-3promoter::mcherry] Figure 1—figure supplement 1D; Figure 4D and E; Figure 4—figure supplement 1B, G and H |
| Strain, strain background (C. elegans) | OK1020 | Kozlova et al., 2019 | n/a | cuIs36 X Figure 2—figure supplement 1D; Figure 2K and L; Video 13 |
| Strain, strain background (C. elegans) | MT23370 | this study | n/a | nIs686 III; lite-1(ce314) gur-3(ok2245) lin-15(n765ts) Figure 2—figure supplement 1E; Figure 3I and L–N; Video 12 |
| Strain, strain background (C. elegans) | VK1241 | CGC | WBStrain00040040 | vkEx1241 [nhx-2p::mCherry::lgg-1 + myo-2p::gfp] Figure 2—figure supplement 1F |
| Strain, strain background (C. elegans) | MT26333 | CGC (not outcrossed); this study (outcrossed) | WBStrain00002254 (not outcrossed); n/a (outcrossed) | sIs11111 [rCesC32F10.8::gfp + pCeh361] 8x outcrossed to N2 Figure 2—figure supplement 2C |
| Strain, strain background (C. elegans) | MT2249 | Horvitz laboratory collection | n/a | egl-47(n1082dm) Figure 3—figure supplement 1A |
| Strain, strain background (C. elegans) | RB850 | CGC | WBStrain00031563 | egl-47(ok677) Figure 3—figure supplement 1B |
| Strain, strain background (C. elegans) | MT21783 | Bhatla and Horvitz, 2015 | n/a | gur-3(ok2245) Figure 3—figure supplement 1C; Figure 3B and G and H |
| Strain, strain background (C. elegans) | RB845 | CGC | WBStrain00031558 | gur-4(ok672) Figure 3—figure supplement 1D |
| Strain, strain background (C. elegans) | FX06169 | Dr. S. Mitani/NBRP | WBVar01474061 (tm6169) | gur-5(tm6169) Figure 3—figure supplement 1E |
| Strain, strain background (C. elegans) | KG1180 | CGC | WBStrain00023485 | lite-1(ce314) 5x outcrossed Figure 3—figure supplement 1F; Figures 3C, G and H; Figure 5A and C; Figure 6J-O |
| Strain, strain background (C. elegans) | MT21793 | Bhatla and Horvitz, 2015 | WBStrain00027622 | lite-1(ce314) gur-3(ok2245) Figure 3—figure supplement 1G; Figure 3D and G and H |
| Strain, strain background (C. elegans) | DA1113 | CGC | WBStrain00005547 | eat-2(ad1113) 2x outcrossed Figure 3—figure supplement 1H and L |
| Strain, strain background (C. elegans) | MT25982 | this study | n/a | eat-2(ad1113); gur-3(ok2245) Figure 3—figure supplement 1H and L |
| Strain, strain background (C. elegans) | MT22899 | this study | n/a | eat-2(ad1113); lite-1(ce314) Figure 3—figure supplement 1I and L |
| Strain, strain background (C. elegans) | MT25983 | this study | n/a | eat-2(ad1113); lite-1(ce314) gur-3(ok2245) Figure 3—figure supplement 1J–1L |
| Strain, strain background (C. elegans) | MT25999 | this study | n/a | eat-2(ad1113); lite-1(ce314) gur-3(ok2245); nIs687 [lite-1promoter::gfp::lite-1; lin-15(+)] nIs687 was outcrossed 8x to MT22499 Figure 3—figure supplement 1K and L |
| Strain, strain background (C. elegans) | MT22499 | this study | n/a | lite-1(ce314) gur-3(ok2245) lin-15AB(n765ts) Used to outcross MT25999 |
| Strain, strain background (C. elegans) | MT25809 | this study | n/a | lite-1(ce314) gur-3(ok2245) lin-15(n765ts); nEx2281 [lite-1promoter::lite-1(+)::gfp; lin-15(+)] Figure 3E and Gand H |
| Strain, strain background (C. elegans) | MT25810 | this study | n/a | lite-1(ce314) gur-3(ok2245) lin-15(n765ts); nEx2157 [gur-3 gDNA; lin-15(+)] Figure 3F-H |
| Strain, strain background (C. elegans) | MT23415 | this study | n/a | nIs686 III; gur-3(ok2245) lin-15(n765ts) Figure 3I, J and M, N |
| Strain, strain background (C. elegans) | MT23417 | this study | n/a | nIs686 III; lite-1(ce314) lin-15(n765ts) Figure 3I, K and M, N |
| Strain, strain background (C. elegans) | MT25631 | this study | n/a | lin-15(n765ts); nIs864 [glr-2promoter::gfp::unc-54 3’ UTR; lin-15(+)] 6x outcrossed Figure 2—figure supplement 1D; Figure 4B and C;Figure 4—figure supplement 1E and F |
| Strain, strain background (C. elegans) | MT25804 | this study | n/a | oxIs322 [myo-2promoter::mcherry::h2b; myo-3promoter::mcherry::h2b] II; nIs310 [nlp-13promoter::gfp] V; nEx2905 Figure 4—figure supplement 1A and C |
| Strain, strain background (C. elegans) | MT25950 | this study | n/a | eat-2(ad1113); nIs865 (integrated transgene derived from nEx2905 [lury-1promoter::ice::sl2::mcherry::unc-54 3’ UTR; ttx-3promoter::mcherry]; outcrossed 8x to N2) Figure 4—figure supplement 1D |
| Strain, strain background (C. elegans) | MT25823 | this study | n/a | lin-15AB(n765ts); nEx2917 [glr-2promoter::hiscl::SL2::mcherry::unc-54 3’ UTR; lin-15(+)] Figure 4H-L |
| Strain, strain background (C. elegans) | MT23192 | this study | n/a | lin-15(n765ts); nIs678 [glr-2promoter::gcamp6s::unc-54 3’ UTR; lin-15(+)] 5x outcrossed. Figure 4M–U, Figure 4—figure supplement 1W; Video 15 |
| Strain, strain background (C. elegans) | MT24110 | this study | n/a | lin-15(n765ts); nIs780 [gur-3promoter::gfp; lin-15(+)] 7x outcrossed to MT8189 Figure 3—figure supplement 1M and N |
| Strain, strain background (C. elegans) | MT23250 | this study | n/a | gur-3(ok2245) lin-15(n765ts); nIs678 Figures 4P, Q, T and U |
| Strain, strain background (C. elegans) | MT23230 | this study | n/a | lite-1(ce314) lin-15(n765ts); nIs678 Figures 4P, R, T and U; Figure 6D–I; Figure 6—figure supplement 1B–D |
| Strain, strain background (C. elegans) | MT23369 | this study | n/a | lite-1(ce314) gur-3(ok2245) lin-15(n765ts); nIs678 Figures 4T and U; Figure 5D and E |
| Strain, strain background (C. elegans) | MT23343 | this study | n/a | unc-13(s69); lin-15(n765ts); nIs678 Figure 4—figure supplement 1I and J |
| Strain, strain background (C. elegans) | MT23410 | this study | n/a | unc-31(u280); lin-15(n765ts); nIs678 Figure 4—figure supplement 1K and L |
| Strain, strain background (C. elegans) | MT25468 | this study | n/a | lite-1(ce314) gur-3(ok2245) lin-15(n765ts); nEx2815 [lury-1promoter::chr2::sl2::mcherry::unc-54 3' UTR] Figure 4V and W |
| Strain, strain background (C. elegans) | CB933 | Horvitz laboratory collection | WBStrain00004216 | unc-17(e245) Figure 4—figure supplement 1M |
| Strain, strain background (C. elegans) | PR1152 | Horvitz laboratory collection | WBStrain00030801 | cha-1(p1152) Figure 4—figure supplement 1N |
| Strain, strain background (C. elegans) | RM509 | CGC | WBStrain00033372 | ric-3(md158) Figure 4—figure supplement 1O |
| Strain, strain background (C. elegans) | CB156 | Brenner, 1974; Takayanagi-Kiya and Jin, 2016 | WBStrain00004114 | unc-25(e156) dnj-17(ju1162) Figure 4—figure supplement 1P |
| Strain, strain background (C. elegans) | MT6308 | CGC | WBStrain00027259 | eat-4(ky5) Figure 4—figure supplement 1Q |
| Strain, strain background (C. elegans) | CB1112 | Horvitz laboratory collection | WBStrain00004246 | cat-2(e1112) Figure 4—figure supplement 1R |
| Strain, strain background (C. elegans) | MT10661 | Horvitz laboratory collection | WBStrain00027379 | tdc-1(n3420) Figure 4—figure supplement 1S |
| Strain, strain background (C. elegans) | MT14984 | Horvitz laboratory collection | WBStrain00027500 | tph-1(n4622) Figure 4—figure supplement 1T |
| Strain, strain background (C. elegans) | MT15434 | Horvitz laboratory collection | WBStrain00027519 | tph-1(mg280) Figure 4—figure supplement 1U |
| Strain, strain background (C. elegans) | MT22280 | Horvitz laboratory collection | MT22280 | ser-4(ok512); mod-1(ok103); ser-1(ok345) ser-7(tm1325) Figure 4—figure supplement 1V |
| Strain, strain background (C. elegans) | MT23606 | this study | n/a | lite-1(ce314) gur-3(ok2245) lin-15AB(n765ts); nEx2144 [flp-15promoter::mcherry::gur-3, lin-15(+)] Figure 5B and C |
| Strain, strain background (C. elegans) | MT23545 | this study | n/a | lite-1(ce314) gur-3(ok2245) lin-15AB(n765ts); nIs678; nEx2144 Figure 5D-H |
| Strain, strain background (C. elegans) | MT26001 | this study | n/a | eat-2(ad1113); lite-1(ce314) gur-3(ok2245) nIs791 X (integrated transgene derived from [nEx2144: flp-15promoter::mcherry::gur-3, lin-15(+)]) Figure 5I |
| Strain, strain background (C. elegans) | MT21421 | Bhatla and Horvitz, 2015 | WBStrain00044110 | nIs569 [flp-15promoter::csp-1b; ges-1promoter::gfp] Figure 6A |
| Strain, strain background (C. elegans) | MT21791 | Bhatla and Horvitz, 2015 | n/a | lite-1(ce314) nIs569 Figure 6B |
Additional files
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Source data 1
Sequences for plasmids generated in this study.
- https://cdn.elifesciences.org/articles/59341/elife-59341-data1-v2.gb.zip
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Source code 1
Matlab scripts.
- https://cdn.elifesciences.org/articles/59341/elife-59341-code1-v2.zip
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Transparent reporting form
- https://cdn.elifesciences.org/articles/59341/elife-59341-transrepform-v2.pdf
