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

Final Report Summary - CELLSTATETRANSITIONS (Capturing transition states associated with lineage decisions in the early mouse embryo)

Introduction
To ensure the faithful development of multicellular organisms, the appropriate number of cells with specific fates have to be specified in uncommitted precursor populations. One of the earliest examples of this process is found in inner cell mass (ICM) of the mammalian preimplantation embryo, in which cells differentiate into epiblast (Epi) and primitive endoderm (PrE). How the proportions of Epi and PrE cells are balanced is not well understood. Transcription factors that specify either lineage are initially co-expressed in individual ICM cells, and their expression patterns becomes mutually exclusive over the course of one to two days in an FGF/MAPK signaling dependent manner (see attached figure). In this project I proposed to model the Epi-versus-PrE fate decision in mouse embryonic stem cells (ESCs) to investigate how the activity of the FGF/MAPK signaling pathway is integrated with that of the transcriptional regulators involved in that fate decision to balance the number of Epi and PrE cells (see attached figure).

Work performed and main scientific results
To establish a tissue culture model to recapitulate the co-expression state of transcriptional regulators specifying the Epi- and the PrE fate, I have developed ES cell lines that carry doxycycline-inducible transgenes encoding fluorescently tagged GATA factors. Addition of doxycycline to these ES cells, which before treatment display molecular features of cells in the Epi compartment such as expression of the Epi-specific transcription factor Nanog, results in the co-expression of Nanog and exogenous GATA factors few hours later (see attached figure), similar to the situation in the ICM of the preimplantation blastocyst. Following removal of doxycycline, this co-expression state is resolved, and 24h later a subset of cells in the culture express endogenous markers of the PrE fate, such as GATA6, in a pattern that is mutually exclusive with Nanog expression at the single cell level (Fig. 1B). This ES cell system therefore recapitulates hallmarks of the fate choice process in the ICM, and allowed me to investigate molecular requirements to make the PrE-like differentiation program accessible, to assess the role of transcription factor levels and to define the influence of signaling activity on PrE-like fate choice, and hence control of the fraction of cells differentiating along the PrE lineage.
First, I tested the influence of different culture conditions on PrE-like differentiation for a given amount of GATA-factor induction, and found that prolonged inhibition of the MAPK signaling pathway before GATA factor expression strongly increase the efficiency of PrE-like differentiation. Secondly, I investigated how GATA factor levels in individual cells influence the decision to embark on PrE-like differentiation. Through time-lapse imaging of the fluorescently tagged GATA-factors in individual cells followed by immunostaining for fate markers I could establish that PrE-like differentiation requires a threshold level of GATA-factor expression in individual cells (see attached figure). Finally, I tested the influence of FGF/MAPK signaling on fate choice by performing differentiation experiments at different signaling levels. This revealed that FGF/MAPK signaling determines the proportion of differentiating cells by setting the threshold of GATA factors required for PrE-like differentiation. Thus, both transcription factor expression levels and signaling together control the proportion of cells differentiating along the PrE-lineage. These results can be formalized in a simple mathematical model of the molecular players underlying fate choice (see attached figure). We validated this model by comparing simulated expression dynamics of the systems’ components with experimentally measured expression dynamics from live reporters generated in the course of this project.

Research output and impact
The work conducted in the course of this project has resulted in three scientific publications and has been presented at a range of national and international conferences.
The project’s impact is both technological and conceptual in nature. Firstly, it pioneers multi-color live-cell imaging as a technique to elucidate the structure of the gene regulatory networks that underlie cell fate choice and balance cell fates in mammalian embryos. Secondly, it uncovered a new principle for signaling in cell fate decisions, which is to control the number of cells in a given lineage by modulating the dynamics of intracellular transcription factor networks. We expect that both the conceptual advances delivered in this project will impact on the research approaches in Developmental Biology, and eventually enhance our understanding, and ability to steer, cell fate decisions in pluripotent cells

Legend for accompanying figure
A Schematic depiction of experimental approach. The decision between the Epi and the PrE fate in the embryo is characterized by a transition from Nanog/GATA co-expression to mutually exclusive expression. This can be recapitulated in ES cells by transient expression of doxycycline-inducible transgenes. B Immunostaining of engineered ES cells shows transition transition from Nanog/GATA co-expression to mutually exclusive expression. C Fluorescence time-traces of GATA4-mCherry expression in engineered cells color-coded for fate choice. A threshold level (black line) of GATA4-mCherry expression separates differentiating from non-differentiating cells. D Architecture of the gene regulatory network that integrates FGF/MAPK signaling and activities of transcriptional regulators in the Epi-versus-PrE fate decision.
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