LAUSR

Cell-fate decisions are governed by comprehensive generegulatory programs. During the preimplantation development, at least two waves of cell fate decisions are made while the cells gradually lose their totipotency (Schrode et al., 2013). The first decision involves the spatial separation of outer-residing trophectoderm (TE) cells from inner cell mass (ICM) in E3.5 mouse blastocyst. The second decision involves gene expression refinements and active cell sorting within the ICM that ultimately results in epiblast (EPI) cells, residing deep within the ICM, and the primitive endoderm (PrE) cells comprising a monolayer of blastocoel-facing cells at the surface of the ICM (Schrode et al., 2013). OCT4, SOX2, and NANOG are master transcription factors (TFs) essential for the formation and maintenance of the pluripotent ICM cells and their in vitro counterparts mouse embryonic stem cells (ESCs). On the other hand, GATA4, GATA6, and SOX17 are master TFs of the PrE cells and their in vitro counterparts extraembryonic endoderm stem cells (XENs). Only naïve ESCs are capable of deriving both primed epiblast stem cells (EpiSCs) and XEN cells in vitro (Cho et al., 2012). Primed EpiSCs cannot derive XEN-like cells, suggesting that ESCs and EpiSCs have different levels of developmental potential (Cho et al., 2012). Understanding the mechanism of pluripotent state transition in vitro provides insights into dynamic control of in vivo developmental transition of embryonic potency while simultaneously preparing for the transition to the somatic lineages. We have investigated the functions of zinc finger protein 281 (ZFP281) in ESC maintenance and ESC-to-EpiSC differentiation (Fidalgo et al., 2011; Fidalgo et al., 2016; Huang et al., 2017). Interestingly, ZFP281 was also reported to be expressed in XEN cells (Wang et al., 2008). However, roles of ZFP281 in the maintenance of XENs and during ESC-toXEN differentiation are elusive. To address this, we firstly examined the expression of ZFP281 and the pluripotency and PrE factors in ESCs, EpiSCs, and XENs. ZFP281 protein is highly abundant at similar levels in pluripotent ESCs and EpiSCs, which is in contrast with a much lower level of ZFP281 protein in XENs (Fig. S1A). However, the mRNA levels of Zfp281 are comparable in all tested cell lines (Fig. S1B), indicating that ZFP281 is regulated at both transcriptional and post-transcriptional levels. Using WT and Zfp281 ESCs (Fidalgo et al., 2011), we investigated the effects of ZFP281 in ESC-to-XEN differentiation following a well-established protocol (Cho et al., 2012) to convert ESCs into stable XEN-like cells (referred to hereafter as chemicalinduced XEN cells, or cXENs, Fig. 1A). Briefly, feeder-free ESCs were treated with retinoic acid (RA) and activin for 2 days, then replated on MEF feeders to further support the culture of cXENs. While RA treatment induced significant differentiation of ESCs at Passage 1 (P1), stellate and refractile XEN-like colonies emerged from both WT and Zfp281 ESCs after replating on MEF feeders (Fig. 1B, P2, white arrows). However, for the cells derived from WT ESCs, after a few days of culture on MEF, compact and domeshape (ESC-like, red arrows in Fig. 1B) colonies reemerged and became dominant at P2 when the cells were confluent. This is probably because that treatment of RA for 2 days, while pushing ESCs to exit pluripotency, is not enough to commit to a XEN fate. In addition, MEF feeders may provide additional factors such as LIF to reestablish pluripotency. In contrast, dome-shaped colonies were rarely seen in Zfp281 ESC-derived cXENs at P2, indicating a more committed XEN fate. When cells were further cultured for one more passage (P3) on feeder-free plates, almost all Zfp281 ESC-derived cXENs showed stellate and highly refractile XEN morphology (Fig. 1B, P3, white arrows), without the need of picking XEN colonies, a necessary step in regular cXEN derivation protocol (Cho et al., 2012). Next, we collected RNAs at P0, P1, P3 (Fig. 1A), when cells were cultured on feed-free plates to avoid RNA contamination from MEF cells, for qRT-PCR analysis. First, we observed that Zfp281 mRNA was activated by RA and activin treatment at P1 in WT cells (Fig. 1C). We also found steady downregulation of pluripotency genes (Oct4, Nanog, Sox2) and