The expression of C. elegans heterochronic miRNAs during larval development, among them
lin-4 and
let-7, has previously been shown to be highly pulsatile, with expression peaking once per larval stage, phase-locked with the molting cycle (Hendriks et al., 2014; Kim et al., 2013, Perales et. al.,2014; Wynsberghe et. al.,2014). While the molecular mechanisms driving this pulsatile expression are unknown, our labs have previously identified two regulators of pulse amplitude. LIN-42/Period is also shown to dampen transcriptional output of miRNAs by negatively regulating the overall duration of transcription within each larval stage (Perales et. al.,2014; Wynsberghe et. al.,2014). Antagonistically,
blmp-1 and
elt-3 null mutants completely suppress
lin-42(lf) phenotypes and BLMP-1 acts as pioneer factor to enhance miRNAs transcription output (Stec et al., 2021). All of the previous approaches to measure the transcriptional dynamics of miRNAs in-vivo have relied on fluorescent transcriptional reporters, limiting temporal and spatial resolution. Here, we present our ongoing efforts to overcome this limitation by monitoring miRNA transcription using MS2/MCP-GFP based RNA-localization. By combining high-resolution long-term imaging with microfluidics (Keil et al., 2017) with extensive image registration, segmentation, tracking and image analysis, we reveal intriguing wave-like spatiotemporal transcriptional patterns of
lin-4 in hypodermal cells as well as vulval precursor cells within larval stages. We also characterize the relationship between lineage descendancy, cell cycle and transcriptional timing among hypodermal and vulval precursor cells. Finally, we measure how these spatiotemporal features of transcription are altered in
lin-42 and
blmp-1 mutants. Our results establish a new approach to measuring live miRNA-dynamics in the C. elegans larva and provide quantitative insights into the complex spatiotemporal regulation of miRNA transcription underlying temporal cell-fate patterning in the C. elegans epidermis.