Plet1 is an epigenetically regulated cell surface protein that provides essential cues to direct trophoblast stem cell differentiation

Abstract

Gene loci that are hypermethylated and repressed in embryonic (ESCs) but hypomethylated and expressed in trophoblast (TSCs) stem cells are very rare and may have particularly important roles in early developmental cell fate decisions, as previously shown for Elf5. Here, we assessed another member of this small group of genes, Placenta Expressed Transcript 1 (Plet1), for its function in establishing trophoblast lineage identity and modulating trophoblast differentiation. We find that Plet1 is tightly repressed by DNA methylation in ESCs but expressed on the cell surface of TSCs and trophoblast giant cells. In hypomethylated ESCs that are prone to acquire some trophoblast characteristics, Plet1 is required to confer a trophoblast-specific gene expression pattern, including up-regulation of Elf5. Plet1 displays an unusual biphasic expression profile during TSC differentiation and thus may be pivotal in balancing trophoblast self-renewal and differentiation. Furthermore, overexpression and CRISPR/Cas9-mediated knockout in TSCs showed that high Plet1 levels favour differentiation towards the trophoblast giant cell lineage, whereas lack of Plet1 preferentially induces syncytiotrophoblast formation. Thus, the endogenous dynamics of Plet1 expression establish important patterning cues within the trophoblast compartment by promoting differentiation towards the syncytiotrophoblast or giant cell pathway in Plet1-low and Plet1-high cells, respectively.

The role of DNA methylation in human trophoblast differentiation

“…The impact of DNA methylation on trophoblast development begins at approximately day 5 post fertilization when the morula splits into the hypomethylated trophectoderm (that gives rise to the trophoblast populations of the placenta) and hypermethylated inner cell mass (that gives rise to the embryo proper) [6]. Segregation of murine TSC from embryonic and extra-embryonic populations (e.g., embryonic stem cells (ESC) and extraembryonic endoderm XEN cells) is associated with distinct methylation signatures in key genes including caudal type homeobox 2 (Cdx2), eomesodermin (Eomes), E74 like ETS transcription factor 5 (Elf5), placenta expressed transcript 1 (Plet1) and transcription factor AP-2, gamma (Tcfap2c), and these methylation changes can be correlated with transcriptional changes between these lineages [6][7][8]. In contrast, the role of DNA methylation in regulating murine trophoblast differentiation has been explored far less [9].…”

“…human embryonic stem cells, neuronal stem cells, mesenchymal stem cells), and it is unclear how they relate to TSC differentiation, which stems from the hypomethylated trophectoderm [20]. In murine TSC hypomethylation of Elf5 and Plet1 promoters contribute to establishing the TSC state and allowing TSC self-renewal [6]. Whether similar DNA methylation events influence human TSC function and differentiation is not yet known however it has been previously reported that that there is a degree of differential methylation between promoter regions of ELF5 between human cytotrophoblasts (3.4% methylated) and EVTs (9.1% methylated) [13].…”

Cellular Localization and Regulation of Expression of the PLET1 Gene in Porcine Placenta

“…A recent report indicates that mouse Plet1 is an important regulator in directing trophoblast cell differentiation. Trophoblast cells with high Plet1 levels preferentially differentiate into cells including spongiotrophoblast [30]. The porcine placenta trophoblast layer is functionally analogous to the trophoblast cells of the labyrinth and spongiotrophoblast layers, thus suggesting a possible conserved role of PLET1 in mediating the pig trophoblast cell differentiation.…”

“…A recent report revealed that mouse Plet1 was repressed in embryonic stem cells caused by DNA methylation but expressed in trophoblast stem cells and trophoblast giant cells. Overexpression of Plet1 could promote the differentiation towards trophoblast giant cell (and/or spongiotrophoblast), but silencing of Plet1was found to induce syncytiontrophoblast formation [30]. These findings suggest an important role of PLET1 in trophoblast cell lineage determination and placentation.…”

Defining totipotency using criteria of increasing stringency

“…To read out fate conversion using flow cytometry, we immunostained for two TSC-specific cell surface markers, CD40 and Plet1. We also established an Elf5-2A-mCherry reporter ESC line by targeting 2A-mCherry to the C-terminus of the endogenous Elf5 locus 41,42,20,22 (Figure 2C). We then switched L-EPSCs, D-EPSCs and ESCs to TSC medium (with Fgf4 and Heparin) with or without tetraycycline (+/-4OH) and cultured the cells for 6 days (6d) before analyzing TSC-marker expression.…”