Introduction
Cell fate decisions refer to the processes by which cells acquire specific properties or functions. This concept is broader than mere differentiation—where undifferentiated cells mature into defined lineages—and also encompasses phenomena such as programmed cell death and changes in cell migration or positioning. Even among a population of cells with identical genomes and in the same environment, variability in gene expression levels (heterogeneity) is observed. This transient fluctuation in gene expression is suggested to influence the eventual fate of cells. One mechanism that generates such heterogeneity is transcriptional bursting. Transcriptional bursting is the phenomenon whereby a gene’s transcriptional activity switches stochastically between an ON state (active RNA synthesis) and an OFF state (little or no RNA synthesis). Because each gene typically exists in only one or two copies per cell, the fluctuations in RNA production resulting from these ON/OFF transitions can lead to significant variability in expression levels among cells (see Figure 1). Recent studies have drawn attention to the idea that stochastic transcriptional activity may underlie deterministic developmental processes.
Figure 1. Transcriptional bursting and the resultant heterogeneity in gene expression. (A) Schematic illustration of transcriptional bursting. (B) Temporal fluctuations in mRNA levels with and without bursting. (C) Differential gene expression between alleles and among cells as a consequence of transcriptional bursting.
Background
In the mouse early embryo, the inner cell mass (ICM) consists of undifferentiated cells that will later give rise to the epiblast (forming the embryo proper) and the primitive endoderm (contributing to extraembryonic tissues). In the early stages of development, all cells in the ICM co-express transcription factors such as NANOG, which is associated with epiblast differentiation, and GATA6, which is associated with ICM differentiation. As development proceeds, cells in the ICM begin to diverge, with some exhibiting high NANOG expression and others high GATA6 expression—forming a “salt-and-pepper” mosaic pattern. Ultimately, NANOG-positive cells tend to become epiblast, while GATA6-positive cells become primitive endoderm (see Figure 2). Although this differentiation process appears random at first glance, evidence indicates that ICM cells with high NANOG levels are more likely to develop into epiblast. Moreover, signals such as FGF4 secreted from NANOG-high cells can induce neighboring cells toward the ICM fate, ultimately leading to the sorting of distinct cell types. Thus, the heterogeneity in gene expression (i.e., the variability in NANOG and GATA6 levels among cells) within the ICM is considered a key factor influencing cell fate decisions (see Figure 2).
Figure 2. Heterogeneity in gene expression levels during early mouse development.
However, the mechanisms underlying the initial variability in NANOG and GATA6 expression within the ICM remain largely unresolved. Recent studies have reported that, in both mouse early embryos and ES cells, the Nanog gene is sometimes transcribed from only one allele. This phenomenon implies that Nanog exhibits pronounced transcriptional bursting, which may contribute to the observed cell-to-cell heterogeneity in NANOG expression. Our previous studies in mouse ES cells have demonstrated that intrinsic noise arising from transcriptional bursting significantly contributes to the variability in NANOG expression (Ochiai et al., Sci Rep, 2014; Ochiai et al., Sci Adv, 2020). On the other hand, there has been little direct investigation into the extent of transcriptional bursting in ICM cells and how it influences cell fate selection. Analyzing the spatiotemporal dynamics of transcription within ICM cells—and its role in the differentiation process toward epiblast or primitive endoderm—is therefore a critical, yet unresolved, issue.
Research Objectives
- To elucidate the relationship between transcriptional bursting and gene expression heterogeneity in the ICM at a single-cell level. Specifically, we aim to quantify the bursting behavior of key genes (e.g., Nanog and Gata6) in ICM cells and determine how these bursts contribute to the variability in gene expression across cells.
- To determine the impact of transcriptional bursting on cell fate decisions. In other words, we will investigate how the frequency and intensity of transcriptional bursts in ICM cells influence their eventual differentiation into epiblast or primitive endoderm.
Research Approach and Methods
DNA/RNA/IF-seqFISH:
We apply a sequential FISH method that combines DNA-FISH, RNA-FISH, and immunofluorescence (IF) on embryonic tissue. This technique allows us to simultaneously visualize and quantify the mRNA expression levels and the protein expression (including transcription factors) in individual ICM cells. For example, mapping the expression distribution of fate-determining factors such as NANOG and GATA6 at the whole-embryo level provides a high-resolution measure of intercellular heterogeneity. In addition, DNA-seqFISH enables us to capture allele-specific transcription states, thus revealing spatial patterns and fluctuations in transcriptional bursting.
Figure: Schematic diagram of DNA/RNA/IF-seqFISH.
Representative Images from seq-DNA/RNA/IF-FISH: A representative video (created from maximum intensity projections of images acquired during each round of seq-DNA/RNA/IF-FISH) shows how three sets of secondary probes and readout probes are used in three channels to capture the localization of a single RNA, a genomic region, a protein, or post-translational modifications (Ohishi et al., Sci Adv, 2020).
Live Single-Gene Imaging:
Using live-cell imaging, we observe transcription dynamics in real time in early embryonic organoids. Specifically, our STREAMING-tag system (Ohishi et al., Nat Commun, 2022) enables visualization of the nuclear localization and mRNA synthesis events of target genes such as Nanog in ICM-like cells. This approach allows us to measure the frequency (i.e., how often a gene enters an ON state) and burst size (i.e., the number of RNA molecules produced per burst) with high temporal resolution, providing insights into the relationship between transcription dynamics and cell fate decisions.
Diagram of the single-gene imaging technology (STREAMING-tag system) and a video captured using this technique. The STREAMING-tag was applied to the Nanog gene in mouse ES cells, and the video was recorded at 15-second intervals.
Diagram of the single-gene imaging technology (STREAMING-tag system) and a video captured using this technique. The STREAMING-tag was applied to the Nanog gene in mouse ES cells, and the video was recorded at 2-minute intervals.
Expected Outcomes and Significance
Elucidating Probabilistic Determinants of Cell Fate:
By quantifying transcriptional bursting and its contribution to gene expression heterogeneity, this study will reveal the molecular basis of the “fluctuations of fate” in early embryos. Quantitative analysis of the interplay between randomness and determinism in gene expression could expand current deterministic models of development and provide new insights into the principles governing cell fate decisions. In particular, demonstrating the role of transcriptional noise during the salt-and-pepper differentiation of ICM cells may resolve longstanding questions in developmental biology.
Implications for Regenerative Medicine and Developmental Biology:
Understanding how noise in gene expression is regulated during differentiation could have profound implications for stem cell biology. In the future, artificially modulating the frequency or intensity of transcriptional bursts might improve the efficiency of directed differentiation protocols or enable precise control over cell fate—advances that could translate into novel therapeutic strategies for regenerative medicine or disease prevention. Additionally, these insights may help explain developmental anomalies such as early embryonic failures.
Future Directions:
The insights gained from this research will not only deepen our understanding of early embryonic cell fate decisions but may also be applied to later developmental stages and other biological contexts. Moreover, candidate regulators of transcriptional bursting identified in this study could eventually be targeted using gene engineering techniques to manipulate cellular noise, further bridging the gap between basic biology and clinical applications.