CELLSTATETRANSITIONS

Capturing transition states associated with lineage decisions in the early mouse embryo

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 Obiettivo del progetto (Objective)

'During early mouse embryogenesis the cells of the blastocyst’s inner cell mass take a lineage decision to contribute either to the epiblast or the primitive endoderm (PE). The allocation of cells to either lineage depends on the activity of FGF signaling and two gene regulatory networks (GRNs), one centered on the transcription factor Nanog, the other one relying on Gata factors. The two GRNs are initially activated in an overlapping and heterogeneous pattern in the ICM, and have been proposed to compete each other out over time. The dynamics of this competition, and how the transition state between the two lineages, marked by co-expression Gatas and Nanog, is resolved, is not known. Here I propose to address these questions in vitro by recapitulating the competition between the Gata- and Nanog-GRNs through the controlled overexpression of Gata factors. This converts embryonic stem cells (ESCs), which contribute primarily to the epiblast when introduced in chimeras, into extraembryonic endoderm (XEN) cells, which contribute solely to PE derivatives. I will combine fluorescent reporters with this ES-to-XEN transition to ask with which dynamics transitions occur in individual cells, whether they involve heterogeneities at the population level, and how these parameters are controlled by the activity of gene regulatory networks and signaling pathways. I will aim at identifying culture conditions that stabilize the transition state, where cells might be on the brink of being XEN, and therefore akin to ICM. I hypothesize, that under these conditions cells will be endowed with higher developmental potential compared to parental ES cells, and be able to contribute to both epiblast and PE-derived tissues. The results of this project will enhance our understanding of the mechanisms underlying lineage decisions in early development and may uncover more general principles that govern the way in which differentiating cells are specified in a stem cell pool.'

Introduzione (Teaser)

In multicellular organisms cells with different fates come from originally uncommitted precursor populations. An EU project investigated the control of cell differentiation in mouse embryonic stem cells (ESCs).

Descrizione progetto (Article)

Both intracellular transcriptional factors and extracellular signalling pathways contribute to cell differentiation. During early mammalian embryogenesis, the cells of the blastocyst's inner cell mass (ICM) differentiate either into epiblast or to the primitive endoderm (PrE). Initially, individual ICM cells co-express the transcription factors affecting this differentiation. Later, the expression patterns become mutually exclusive, responding to the extracellular signalling.

The EU-funded project CELLSTATETRANSITIONS (Capturing transition states associated with lineage decisions in the early mouse embryo) took a closer look at these events. The project used mouse ESCs as a tissue culture model. The researchers engineered cell lines carrying doxycycline-inducible genes, encoding fluorescently tagged GATA transcription factors. These DNA-binding proteins control differentiation processes in the cell by activating or repressing transcription. The project studied the interplay between the transcriptional regulation and a signalling pathway, involving fibroblast growth factors (FGFs) and mitogen-activated protein kinases (MAPKs).

CELLSTATETRANSITIONS showed how the levels of GATA factor in individual cells influenced the cell fate to undergo PrE-like differentiation. Scientists used time-lapse imaging of the fluorescently tagged GATA factors in individual cells, followed by immunostaining for fate markers. They found that PrE-like differentiation required a threshold level of GATA factor expression in individual cells. Differentiation experiments at different signalling levels revealed that FGF/MAPK signalling determined the proportion of differentiating cells by setting the threshold of GATA factors.

The project demonstrated that both transcription factor expression levels and signalling control the proportion of cells differentiating along the PrE lineage. The team suggested a simple mathematical model to describe the events underlying fate choice and validated this model by comparing simulated expression dynamics with experimentally measured processes. The resulting three scientific publications uncover a new principle for signalling in cell fate decisions, controlling the number of cells in a given lineage. In addition to new scientific understanding, the project pioneered multi-colour live cell imaging elucidating the structure of the gene regulatory networks.