Abstract
ABSTRACTProtocols have been established that direct differentiation of human pluripotent stem cells into a variety of cell types, including the endoderm and its derivatives. This model of differentiation has been useful for investigating the molecular mechanisms that guide human developmental processes. Using a directed differentiation protocol combined with shRNA depletion we sought to understand the role of GATA6 in regulating the earliest switch from pluripotency to definitive endoderm. We reveal that GATA6 depletion during endoderm formation results in apoptosis of nascent endoderm cells, concomitant with a loss of endoderm gene expression. We show by chromatin immunoprecipitation followed by DNA sequencing that GATA6 directly binds to several genes encoding transcription factors that are necessary for endoderm differentiation. Our data support the view that GATA6 is a central regulator of the formation of human definitive endoderm from pluripotent stem cells by directly controlling endoderm gene expression.
Highlights
Studies in zebrafish, frogs, nematodes and mice have identified key growth factors and transcriptional regulators that promote endoderm specification and development of endoderm-derived tissues
We measured steady-state levels of mRNAs encoding diagnostic differentiation markers by real-time quantitative polymerase chain reaction (RT-qPCR) in samples collected from pluripotent H1 human embryonic stem cells or differentiating endoderm at each day after induction
Cumulatively, our results demonstrate that GATA6 acts upstream of GATA4 to regulate expression of genes that ensure the viability of the definitive endoderm
Summary
Frogs, nematodes and mice have identified key growth factors and transcriptional regulators that promote endoderm specification and development of endoderm-derived tissues. In this context, researchers have intensely examined the contribution of the GATA binding protein family. GATA4, GATA5 and GATA6 are the most prominently expressed GATA binding proteins during the development of both extra-embryonic and definitive endoderm cell lineages in mouse embryos (Laverriere et al, 1994; Morrisey et al, 1996). Providing GATA null embryos with a wild-type extra-embryonic endoderm through tetraploid complementation circumvented the lethality, and revealed roles for GATA4 and GATA6 in heart and liver development (Narita et al, 1997; Zhao et al, 2005, 2008; Watt et al, 2007)
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