Inhibitory peptidergic modulation of C. elegans serotonin neurons is gated by T-type calcium channels
- The Helen L. and Martin S. Kimmel Center for Biology and Medicine, Department of Cell Biology, NYU Langone School of Medicine, United States
Figures
Figure 1 with 2 supplements
The n5209 mutation is a potent and specific suppressor of inhibitory neuropeptide signaling.
(A) Micrograph of egl-6(gf) and egl-6(gf) n5209 animals. Eggs are colored for emphasis. Asterisk indicates vulva. (B) Distribution of the developmental stages of eggs laid by wild type, egl-6(gf), …
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Figure 1—source data 1
Raw data of developmental stage of newly laid embryos from Figure 1.
- https://doi.org/10.7554/eLife.22771.003
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Figure 1—source data 2
Time of egg-laying event during tracking.
- https://doi.org/10.7554/eLife.22771.004
Figure 1—figure supplement 1
Anatomy and connectivity of the C. elegans egg-laying circuit.
Arrows denote synaptic connections. Dashed lines denote extrasynaptic inputs. Neurons are green. Sex muscles are red.
Figure 1—figure supplement 2
Histogram of interval times on a natural log scale.
Data replotted from Figure 1F.
Figure 2 with 1 supplement
The n5209 suppressor of peptidergic inhibition affects the T-type calcium channel gene cca-1.
(A) The suppressor mutation, n5209, was mapped to a 2.5 Mb interval on Linkage Group X using a marked egl-6(gf) strain (top). Inset: Gene models within the 2.5 Mb interval. Sequence variants in this …
Figure 2—figure supplement 1
X-linked sequence variants in n5209 and n6122 mutants.
New sequence variants were identified in the independently derived n5209 and n6122 mutants. Variants were called using the Genome Analysis ToolKit (McKenna et al., 2010a; DePristo et al., 2011; Van …
Figure 3
The n5209 suppressor mutation alters cca-1 function.
(A) cca-1(n5209) is a potent suppressor of egl-6(gf) (top; data replotted from Figure 1). One copy of the n5209 allele was sufficient to partially suppress the egl-6(gf) phenotype (bottom). (B) A …
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Figure 3—source data 1
Raw data of developmental stage of newly laid embryos from Figure 3.
- https://doi.org/10.7554/eLife.22771.010
Figure 4 with 1 supplement
The n5209 mutation changes the voltage-dependence of steady-state activation and inactivation of T-type channels.
(A–C) Steady-state activation of wild-type and mutant T-type channels expressed in X. laevis oocytes. (A) Representative trace of wild type Cav3.1, the mammalian homolog of CCA-1. (B) Representative …
Figure 4—figure supplement 1
n5209 mutation shifts the current-voltage relationship of Cav3.1 to more hyperpolarized potentials.
IV curves of wild-type (blue) and mutant (red) channels. N = 10 for wild type; N = 5 for mutant.
Figure 5
T-type Ca2+ channels act in parallel to the GPCR signaling pathway that mediates peptidergic inhibition.
(A) cca-1(n5209) enhanced suppression of the egl-6(gf) phenotype caused by loss of its downstream effector, GOA-1. (B) cca-1(n5209) enhanced the suppression of egl-6(gf) caused by the loss of its …
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Figure 5—source data 1
Raw data of developmental stage of newly laid embryos from Figure 5.
- https://doi.org/10.7554/eLife.22771.014
Figure 6 with 2 supplements
The T-type Ca2+ channel CCA-1 acts in the HSNs to suppress excess neuropeptide inhibition.
(A) A cca-1 fosmid reporter (green) is expressed in many head neurons. (B) A cca-1 fosmid reporter (green) is expressed in the egg-laying system and is co-expressed in the HSNs with an egl-6 …
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Figure 6—source data 1
Number of GFP+ VCs with the cca-1 fosmid.
- https://doi.org/10.7554/eLife.22771.016
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Figure 6—source data 2
Raw data of developmental stage of newly laid embryos from Figure 6.
- https://doi.org/10.7554/eLife.22771.017
Figure 6—figure supplement 1
CCA-1 expression in the VCs is dependent on the homeodomain-containing transcription factor lin-39.
(A) Wild-type animals expressing a 3 kb cca-1 promoter driving GFP. Most animals expressed Promcca-1::GFP in both VC4 and VC5. No animals expressed Promcca-1::GFP in HSNs. Asterisk denotes vulva. (B)…
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Figure 6—figure supplement 1—source data 1
Number of GFP+ VCs with the short cca-1 promoter.
- https://doi.org/10.7554/eLife.22771.019
Figure 6—figure supplement 2
Knockdown of CCA-1 in wild-type HSNs weakly affects egg-laying.
HSN-specific knockdown of CCA-1 in HSNs in wild-type animals. Shown are pooled data from two strains outcrossed in parallel from egl-6(gf) cca-1(n5209); cca-1(RNAi) in HSNs.
Figure 7 with 1 supplement
Re-tuned channels restore neural activity to inhibited serotonin neurons.
(A) Representative △F/F traces of spontaneous somatic calcium transients in HSNs of partially restrained adult hermaphrodites. (B) Cumulative △F/F of wild-type and mutant HSN somas. Values for …
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Figure 7—source code 1
- https://doi.org/10.7554/eLife.22771.022
Figure 7—figure supplement 1
Excess peptidergic inhibition onto HSNs does not significantly alter spontaneous vulval muscle activity.
(A) Representative ΔF/F traces of spontaneous vulval muscle calcium transients in live, partially restrained adult hermaphrodites. (B) Cumulative ΔF/F from wild-type and mutant vulval muscle calcium …
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Additional files
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Supplementary file 1
List of all p-values.
- https://doi.org/10.7554/eLife.22771.024
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Supplementary file 2
Plasmid sequences.
- https://doi.org/10.7554/eLife.22771.025
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Supplementary file 3
List of strains used in this study.
- https://doi.org/10.7554/eLife.22771.026
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Supplementary file 4
List of primers and plasmids used in this study.
- https://doi.org/10.7554/eLife.22771.027
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Supplementary file 5
Sequence of RNAi seed.
- https://doi.org/10.7554/eLife.22771.028
