Gata3 regulates trophoblast development downstream of Tead4 and in parallel to Cdx2

Abstract

The mouse blastocyst and stem cells derived from its tissue lineages provide a unique genetic system for examining the establishment and loss of pluripotency. The transcription factor Cdx2 plays a central role by repressing pluripotency genes, such as Oct4, and promoting extraembryonic trophoblast fate at the blastocyst stage. However, genetic evidence has suggested that Cdx2 does not work alone in the trophoblast lineage. We have used bioinformatic and functional genomic strategies to identify the transcription factor Gata3 as a trophoblast factor. We show Gata3 to be capable of inducing trophoblast fate in embryonic stem cells and driving trophoblast differentiation in trophoblast stem cells. In addition, Cdx2 is not required for Gata3-induced expression of a subset of trophoblast genes in embryonic stem cells. We show that Gata3 is coexpressed with Cdx2 in the blastocyst, but this does not depend on Cdx2. In the embryo, expression of Gata3, like that of Cdx2, depends on Tead4, and the expression of both factors becomes restricted to trophoblast by a mechanism that does not initially rely on Oct4. These observations suggest that Gata3 and Cdx2 can act in parallel pathways downstream of Tead4 to induce the expression of common and independent targets in the trophoblast lineage, whereas Oct4 is required for continued repression of trophoblast fate in the embryonic lineage.

Cdx2Efficiently Induces Trophoblast Stem-Like Cells in Naïve, but Not Primed, Pluripotent Stem Cells

“…In all of the ESC-derived TSC-like cells, TSC genes were detected at levels comparable with TSCs (5/5 subclones; Fig. 1H), indicating that the ESC-derived TSC-like cells expressed TSC markers, consistent with prior reports [16,22]. As expected, expression of TSC genes was barely detectable in differentiated ESCs lacking Cdx2ER (Fig.…”

“…We first confirmed that ESCs had acquired TSC morphology after Cdx2 overexpression, as previously demonstrated [16,22]. Under conditions that support ESC selfrenewal, ESCs grow as small domed colonies (Fig.…”

“…ECCs were maintained in DMEM with 15% FBS, l-glutamine, and penicillin/streptomycin. Linearized pCAG-Cdx2ERT2-ires-puro r construct was introduced into cells, and stably transformed colonies were selected and screened as previously described [22]. The Cdx2 overexpression assay was carried out by harvesting confluent wells of pluripotent cells and seeding at a 1:100 split ratio.…”

“…Subsequently, we attempted to derive TSCs by treating multiple Cdx2ER-expressing ESC and EpiSC subclones with Tx in TSC medium on MEFs for 6 days (Fig. 1A, K), as previously described [16,22]. As a negative control, we (Fig.…”

“…For example, ESCs can be converted to TSCs by overexpressing the TE-specific transcription factor CDX2 in TSC medium [16] and by other means [17][18][19][20][21]. Importantly, overexpression of Cdx2 in ESCs leads to TSC-like cells with highly similar morphology, developmental potential, and gene expression as embryoderived TSCs [16,22,23]. Similarly, TSCs can be converted to ESC-like iPSC by overexpressing Oct4 [24,25].…”