Evaluating Gene Expression Dynamics Using Pairwise RNA FISH Data

Recently, a novel approach has been developed to study gene expression in single cells with high time resolution using RNA Fluorescent In Situ Hybridization (FISH). The technique allows individual mRNAs to be counted with high accuracy in wild-type cells, but requires cells to be fixed; thus, each cell provides only a “snapshot” of gene expression. Here we show how and when RNA FISH data on pairs of genes can be used to reconstruct real-time dynamics from a collection of such snapshots. Using maximum-likelihood parameter estimation on synthetically generated, noisy FISH data, we show that dynamical programs of gene expression, such as cycles (e.g., the cell cycle) or switches between discrete states, can be accurately reconstructed. In the limit that mRNAs are produced in short-lived bursts, binary thresholding of the FISH data provides a robust way of reconstructing dynamics. In this regime, prior knowledge of the type of dynamics – cycle versus switch – is generally required and additional constraints, e.g., from triplet FISH measurements, may also be needed to fully constrain all parameters. As a demonstration, we apply the thresholding method to RNA FISH data obtained from single, unsynchronized cells of Saccharomyces cerevisiae. Our results support the existence of metabolic cycles and provide an estimate of global gene-expression noise. The approach to FISH data presented here can be applied in general to reconstruct dynamics from snapshots of pairs of correlated quantities including, for example, protein concentrations obtained from immunofluorescence assays.Author Summary: Programs of gene expression lie at the heart of how cells regulate their internal processes. Some dynamical gene-expression programs, such as the cell cycle, are well known and studied, others, such as metabolic cycles, have only recently been recognized, and many other dynamical programs including switches are likely to be discovered. Traditional bulk studies typically fail to resolve such cycles or switches, because individual cells are out-of-phase with each other. On the other hand, standard techniques for studying single cells are limited in time resolution and scope. RNA Fluorescent In Situ Hybridization (FISH) is a single-cell technique that offers both high time-resolution and precise quantification of mRNA molecules, but requires fixed cells. We have explored how, when, and with what prior information FISH snapshots of pairs of genes can be used to accurately reconstruct gene-expression dynamics. The technique can be readily implemented, and is broadly applicable from bacteria to mammals. We lay out a principled and practical approach to extracting biological information from RNA FISH data to reveal new information about the dynamics of living organisms.