MONITTR reveals the induced synthesis of hsp-16.41 transcripts in embryos and larvae upon heat shock. (A) Fluorescent images showing the nascent RNA of hsp-16.41 at various embryonic stages. Embryos were imaged immediately after heat shock at 33°C for 30 min. BFP::LMN-1 (blue) labels the nuclear membrane. Arrowheads: nascent RNA labeled by MCP::GFP. The boxed regions are magnified (2.8×). Scale bars: 5 μm. (B) Quantification of the number of active alleles of hsp-16.41 in A. For each group, n = 10 embryos were analyzed. The number of cells quantified were indicated on top of the bar for each strain. (C and D) Labeling of hsp-16.41 nascent transcripts by MCP::GFP and smFISH using probes against MS2 sequence. RNA FISH probes against DapB were used as the negative control. Nuclei were stained by DAPI (blue). Embryos were collected immediately after heat shock at 33°C for 30 min. Arrowheads: mRNA spots. The boxed region is magnified (2×). Scale bars: 5 μm. n represents the number of embryos. All values are displayed as mean ± SEM. ****P < 0.0001 (two-tailed unpaired Student’s t test). (E and F) Representative images and quantification of hsp-16.41 transcripts response to heat shock in PVD neurons. 1-day-old adult animals were imaged immediately after heat shock at 33°C for 30 min. Arrowheads: nascent RNA. Scale bars: 5 μm. 25 PVD neurons from 25 animals were analyzed. (G) Representative images to show the labeling of nascent transcripts by MCP::GFP at hsp-16.41 loci in the intestine cells of C. elegans larvae. Animals at different stages were imaged immediately after heat shock at 33°C for 30 min. BFP::LMN-1 labels the nuclear membrane. Arrowheads: nascent RNA. For L1 group, the boxed regions are magnified (3×). For the L2 group, the boxed regions are magnified (3.2×). For L3 and L4 groups, the boxed regions are magnified (2.8×). Scale bars: 5 μm. (H) Quantification of the number of active alleles of hsp-16.41 in E. The number of cells analyzed is indicated above each bar.