Abstract
During mammalian preimplantation development, the cells of the blastocyst's inner cell mass differentiate into the epiblast and primitive endoderm lineages, which give rise to the fetus and extra-embryonic tissues, respectively. Extra-embryonic endoderm (XEN) differentiation can be modeled in vitro by induced expression of GATA transcription factors in mouse embryonic stem cells. Here, we use this GATA-inducible system to quantitatively monitor the dynamics of global proteomic changes during the early stages of this differentiation event and also investigate the fully differentiated phenotype, as represented by embryo-derived XEN cells. Using mass spectrometry-based quantitative proteomic profiling with multivariate data analysis tools, we reproducibly quantified 2,336 proteins across three biological replicates and have identified clusters of proteins characterized by distinct, dynamic temporal abundance profiles. We first used this approach to highlight novel marker candidates of the pluripotent state and XEN differentiation. Through functional annotation enrichment analysis, we have shown that the downregulation of chromatin-modifying enzymes, the reorganization of membrane trafficking machinery, and the breakdown of cell-cell adhesion are successive steps of the extra-embryonic differentiation process. Thus, applying a range of sophisticated clustering approaches to a time-resolved proteomic dataset has allowed the elucidation of complex biological processes which characterize stem cell differentiation and could establish a general paradigm for the investigation of these processes.
Highlights
The specification of different cell types during development is a complex process that is organized in a hierarchical fashion
We have made use of the fact that Embryonic Stem (ES) cells can be differentiated in an efficient and synchronous manner towards Primitive Endoderm (PrE)-like lineages through the induced expression of GATA transcription factors [11, 12], and used this system to investigate with high temporal resolution the dynamic changes that occur in the cell’s proteome as they alter their fate and function
Isobaric labeling and mass spectrometry-based proteomic profiling coupled with multivariate data analysis techniques allowed us to capture the dynamic and sequential response of over 2,000 proteins following GATA4 induction
Summary
The specification of different cell types during development is a complex process that is organized in a hierarchical fashion. At embryonic day E3.0, the mouse embryo is comprised of an outer epithelial layer, the trophectoderm, that will give rise to the placenta and an inner cell mass (ICM) which will be subdivided into the Primitive Endoderm (PrE) and the Epiblast (Epi). All cells in the ICM co-express transcription factors for the Epi fate, such as OCT4, SOX2 and NANOG, together with the transcriptional regulator GATA6 that promotes PrE specification and is a marker for this fate at later stages. In addition to these differences in transcription factor expression, the PrE and the Epi are morphologically distinct: while the Epi prior to implantation has mesenchymal character, the PrE initially forms an epithelium lining the epiblast. The PrE further differentiates into the epithelial visceral endoderm, a tissue surrounding the post-implantation epiblast, and the parietal endoderm, which deposits the extracellular matrix of Reichert’s membrane [4, 5]
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