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A repressive regulatory cascade shapes temporal patterning of activity-regulated gene expression in a defined sensory neuron type

Samuel G. BatesiD, Nathan Harris, Stephen NurrishiD, Piali SenguptaiD

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Abstract

Long-term neuronal plasticity is driven by activity-regulated gene (ARG) expression programs that encode stimulus features in a neuron type-specific manner 1-6. ARG programs are typically characterized by rapid induction of immediate early genes (IEGs) without requiring new protein synthesis, followed by expression of secondary response genes regulated by IEG-encoded transcription factors 2,5,7-14. However, the molecular mechanisms that pattern these programs in specific neuron types in vivo in response to physiological stimuli are not fully described. We previously showed that temperature regulates an ARG program in the C. elegans AFD thermosensory neuron pair to drive behavioral plasticity 3,15,16. By profiling AFD following temperature upshifts of varying duration, here we show that ARGs in this neuron exhibit distinct temporal trajectories. Notably, rapidly induced genes do not include known IEGs but are enriched for molecules implicated in signal transduction and navigation. Both rapid and delayed ARG expression require the CMK-1 CaMKI kinase and CRH-1 CREB transcription factor, with CRH-1 acting at both early and late stages. We further define a temporal regulatory cascade in which CREB-dependent rapid induction of the RCAN-1 calcineurin regulator acts in parallel with the MEF-2 transcription factor to repress premature expression of a delayed ARG. Subsequent downregulation of RCAN-1 likely enables CRH-1-dependent ARG expression at later stages. Our results demonstrate that in addition to classical gene-activating transcriptional cascades, ARG-controlled repressive mechanisms also operate to precisely shape the temporal dynamics of an ARG cascade in a sensory neuron type in vivo, and suggest that distinct cell type-specific regulatory pathways operate to drive ARG expression programs across neuron types.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 3 (29 Jul 2026) · CC BY 4.0

TRAP-seq of C. elegans AFD neurons reveals three temperature-duration expression modules; CREB-driven RCAN-1 acts in parallel with MEF-2 to repress premature expression of a delayed gene, ins-39.

How activity-regulated gene programs are patterned in defined neuron types in vivo under physiological stimuli is poorly described, and the classical model centers on immediate early transcription factors activating later genes. Here, AFD rapid genes lack canonical IEGs, and a feedback repressive pathway sets the timing of a delayed gene.

Key results

  1. ShownUpregulated genes in AFD fall into three temporal co-expression modules: rapid transient, rapid sustained, and delayed. Their expression reflects how long the animal has experienced warming.

    DPGP clustering of genes upregulated at 1 hr and 4 hr at 25°C (after growth at 15°C) gave three clusters. Cluster 1 was largest, cluster 3 smallest with significant induction only at 4 hr. Data from 4 independent TRAP-seq experiments. · The duration of temperature experience is…; Figure legends; Statistical analyses

  2. ShownRapidly induced AFD genes include no canonical IEGs. They are enriched for signaling, temperature-response and taxis functions, and actin-myosin genes sit mainly in the rapid sustained cluster.

    Canonical IEGs fos-1, jun-1 and egr1-related genes were absent from rapidly induced genes. GO terms for signaling and taxis were spread across clusters 1 and 2; actin-myosin terms were mostly cluster 2. · The duration of temperature experience is…

  3. ShownCMK-1 and CRH-1 support induction of a subset of rapid genes (rcan-1, cebp-1 at 4 hr) and of the delayed gene ins-39. CRH-1 stays phosphorylated and is needed at both early and late phases of warming.

    Auxin-mediated CRH-1 depletion during the whole 24 hr course or the final ~20 hr abolished ins-39 induction; the first 4 hr alone also reduced it (4 independent experiments). Phosphorylation sensor was nuclear at 4 hr and overnight (n>150 AFD cells per condition, 8 independent experiments). · CRH-1 CREB acts both early and late to regulate…; Figure legends

  4. ShownRCAN-1 and MEF-2 act in parallel to block premature CRH-1-dependent ins-39 expression at early time points, and CRH-1 phosphorylation is also enhanced in rcan-1 mutants.

    ins-39 reporter rose slightly in rcan-1 at 4 hr, was unchanged in mef-2 alone, and was significantly higher in mef-2; rcan-1 double mutants; this was abolished by crh-1 mutation. CRH-1 sensor signal was enhanced in rcan-1 (10 independent experiments for reporter; 4 for sensor). · Delayed upregulation of ins-39 expression is…; Figure legends; SUPPLEMENTAL FIGURE LEGENDS

  5. SuggestedLoss of rcan-1 lowers the AFD response threshold (T*AFD) 4 hr after a temperature upshift, and AFD-specific rcan-1 expression rescues it, supporting a role for RCAN-1 timing in tuning plasticity.

    rcan-1 mutants had significantly lower T*AFD at 4 hr after upshift, rescued by AFD-specific rcan-1 expression; after overnight growth at 15°C T*AFD shifted warmer (3 independent experiments, one-way ANOVA with Dunnett's correction). · Mutations in rcan-1 alter AFD thermosensory…; Figure legends; SUPPLEMENTAL FIGURE LEGENDS

Methods, data and reproducibility

Ribosome-associated RNA from AFD was profiled by TRAP-seq after 15°C to 25°C upshifts, and genes were clustered by trajectory. Candidate regulators were tested with endogenous SL2 reporters, mutants, a CRH-1 phosphorylation sensor, auxin-inducible degradation and GCaMP imaging.

  • AFD TRAP-seq at 15°C, 1 hr and 4 hr at 25°C, compared with whole-animal lysates · C. elegans · n = 4 independent experiments · PRJNA1503593
  • Fluorescent transcriptional/translational reporter imaging in AFD soma across genotypes (crh-1, cmk-1, rcan-1, mef-2, cebp-1, tax-6) · C. elegans · n = 2 to 10 independent experiments depending on figure
  • CRH-1 phosphorylation sensor (KID-KIX) localization in AFD · C. elegans · n = n>150 AFD cells per condition; 8 independent experiments
  • GCaMP6s calcium imaging of AFD response threshold during a linear temperature ramp · C. elegans · n = 3 independent experiments
Methods & tools
TRAP-seq, DESeq2, STAR, cutadapt, DPGP_cluster (Dirichlet process Gaussian process mixture), clusterProfiler, GOSemSim, CRISPR/Cas9 genome editing, SL2::H2B::gfp11 split-GFP reporters, auxin-inducible degron, GCaMP6s, MATLAB
Software & environment
DPGP_cluster run in Python2; DESeq2, clusterProfiler and GOSemSim in R; custom image and calcium analysis in MATLAB. No package versions given.
Reproducibility gaps
Strain and gene-editing reagent lists are in supplementary Excel tables; the GEO TRAP-seq record is cited only as a BioProject accession.
Limitations (4)
  • TRAP-seq measures mRNA association with ribosomes, so altered levels may reflect transcriptional and non-transcriptional regulation; reporter and protein dynamics sometimes diverge from TRAP-seq trajectories. · stated by the authors
  • After early-window auxin removal, the authors cannot exclude that CRH-1 levels were incompletely restored, which complicates the interpretation of early-phase CRH-1 requirement. · stated by the authors
  • The model that RCAN-1 acts via TAX-6 calcineurin and that MEF-2 competes with CRH-1 or modulates chromatin is inferred from genetics; no direct biochemical or occupancy tests are reported. · noted in this summary
  • The repressive cascade is dissected mainly for one delayed gene (ins-39) and a few representative reporters; whether it generalizes to the delayed cluster is untested. Downregulated genes were not analyzed. · noted in this summary

Read this if you study activity-regulated transcription, CREB/calcineurin signaling, or how single neuron types time gene expression programs in vivo.

Citations
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