High-throughput single-molecule FISH to calibrate in-vivo transcriptional kinetics of developmental timing genes in C. elegans

Spatiotemporal patterns of gene expression are fundamental to every developmental program. The resulting gene expression domains are traditionally characterized by their levels of gene products. However, in vivo transcriptional dynamics are known to be highly dynamic, often stochastically fluctuating between periods of quiescence and periods of pronounced transcriptionally activity¹. To fully understand how patterns of gene expression are established and maintained during development, studies of in-vivo transcription kinetics are required.

Our teams has recently developed technologies that allow us to visualize and analyze transcription dynamics of microRNA (miRNA) genes in developing C. elegans larva in real-time using the MS2-MCP-GFP tethering system². The MS2-MCP-GFP system, exploits the natural affinity between RNA loops derived from MS2 bacteriophage to function as an RNA epitope tag that can be bound by a co-expressed MS2 coat protein fused to green fluorescent protein (MCP-GFP). By genetically encoding the MS2 hairpins within a gene of interest, nascent RNAs can be directly imaged in living cells through their ability to be concentrated at the sites of RNA synthesis. The exceptional temporal resolution of this system can then be used to determine how specific cis-regulatory sequences control transcriptional burst size, frequency and duration³.

Using this system, we have previously shown that miRNA genes are described in precisely-timed short transcriptional episodes, or pulses, within each post-embryonic stage of C. elegans development². The goal of this internship is to combine live-imaging of developmental gene transcription with high-throughput single-molecule fluorescent in situ hybridization (FISH; smFISH) in developing C. elegans larvae to obtain a single-molecule view of this intriguing transcriptional kinetics. We are particularly interested in precise estimates of the number of actively transcribing RNA pol II molecules at the loci of the miRNA lin-4, as well as the total number of miRNAs produced during each transcriptional episode. lin-4 is a highly conserved miRNA whose transcriptional dosage is modulated by a rewired circadian-like gene regulatory network in C. elegans. Once the analysis is performed for WT animals, we will analyze the impact of specific deletions in regulatory sequences upstream of the lin-4 gene as well as mutations in the circadian network driving lin-4 transcription.

miRNA dysregulation influences critical molecular pathways involved in tumor progression, invasion, angiogenesis and metastasis in a wide range of cancer types. The quantitative analyses proposed in this internship are an important step towards understanding their regulation in normal development and pathology. Candidates for this internship should have a strong motivation for interdisciplinary research, a background in developmental biology, cell biology, biophysics or a related field and most important a passion to see and study life in action. Experience with live microscopy, microfluidics, immunostaining or single-molecule techniques is a plus.

1. What Is a Transcriptional Burst? Trends in Genetics (2020) Tunnacliffe E, Chubb JR ; https://doi.org/10.1016/j.tig.2020.01.003 ↩︎
2. Circadian rhythm orthologs drive pulses of heterochronic miRNA transcription in C. elegans. bioRxiv (2023), Developmental Cell; Kinney B*, Sahu S*, et al.; https://doi.org/10.1016/j.devcel.2023.08.006 ↩︎
3. Quantitative Imaging of Transcription in Living Drosophila Embryos Links Polymerase Activity to Patterning. Current Biology (2013) Garcia HG, et al.; https://doi.org/10.1016/j.cub.2013.08.054 ↩︎

Team references

1. Circadian rhythm orthologs drive pulses of heterochronic miRNA transcription in C. elegans. bioRxiv (2023), Developmental Cell; Kinney B*, Sahu S*, et al.; https://doi.org/10.1016/j.devcel.2023.08.006
2. An Epigenetic Priming Mechanism Mediated by Nutrient Sensing Regulates Transcriptional Output during C. elegans Development. Current Biology (2021) Stec N, et al.; https://doi.org/10.1016/j.cub.2020.11.060
3. HLH-2/E2A Expression Links Stochastic and Deterministic Elements of a Cell Fate Decision during C. elegans Gonadogenesis. Current Biology (2019) Attner MA*, Keil W*, et al.; https://doi.org/10.1016/j.cub.2019.07.062
4. Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics. Developmental Cell (2017) Keil W, et al. ; https://doi.org/10.1016/j.devcel.2016.11.022