MONITTR allows real-time tracking of the dynamic changes of adh-1 transcription in wild-type, nhr-49 and hlh-30 mutant animals. (A) Snapshots of time-lapse recordings of DualTag-based imaging to report the transcriptional activity of adh-1 upon fasting. L4 stage animals were imaged. All images are maximum intensity projections from z-stacks. Arrowheads: nascent RNA spots. Scale bars: 5 μm. (B) Quantification of the tissue-wide transcription activity of adh-1 in the strains shown in A, which were defined by the percentage of nuclei with RNA spots. n = 9 animals for WT, n = 16 animals for nhr-49(zac598) and n = 13 animals for hlh-30(tm1978) were measured, including the ones showed in A. (C) Heatmaps to show the nuclear-wide transcriptional activity of adh-1 in different strains showed in B, which were defined by the total intensity of MCP::GFP spots and the number of transcriptionally active alleles per nucleus. n = 254 nuclei for WT, n = 377 nuclei for nhr-49(zac598) and n = 310 nuclei for hlh-30(tm1978) were quantified. (D) Heatmaps showing the allele-wide transcriptional activity defined by MCP::GFP spots in each allele being in burst or pause of adh-1 in A. n = 30 alleles for WT, n = 30 alleles for nhr-49(zac598) and n = 39 alleles for hlh-30(tm1978) were quantified. (E) Representative quantifications to show the transcriptional dynamics of single alleles upon fasting as shown in D. (F) Quantification of the total burst time or total pause time in different strains showed in D. n represents the number of alleles. (G) Quantifications of single burst duration (left) or single pause duration (right) in different strains as shown in D, n represents the number of bursts or pauses. (H) Quantification of burst amplitude is defined by the maximum total intensity of each MCP::GFP-labeled spot in a single burst in D. n represents the number of bursts. ns: not significant. *P < 0.05 (one-way ANOVA with the Tukey correction).