Trophoblast cells initiate the first developmental lineage in the mammalian conceptus and are thus responsible for embryo implantation and placental development, the defining characteristic of eutherians. There is an ever-increasing list of growth factors and their receptors, signaling molecules, transcription factors, and proteases that have been shown to regulate various stages of trophoblast development. We demonstrate here that TS cell self renewal requires Ets2 activity. Inactivation of Ets2 caused placental insufficiency including a smaller ectoplacental cone and an absence of ExE, the area containing TS cells proliferating under the influence of FGF4 produced by embryonic ectoderm (Kunath et al., 2004
; Tanaka et al., 1998
). We showed evidence of deficient isolation of Ets2db1/db1
TS cells and the selective disadvantage of Ets2db2/db2
TS cells under both high and low density growth conditions, indicating a requirement of Ets2 activity for continuous TS proliferation. TS cells express at least 18 different members of the Ets family of transcription factors (unpublished data). However, deficiency of a single member of the family, Ets2, caused defective self renewal of TS cells, which is not complemented by any other Ets member. In contrast, Ets2 deficient ES cells have no apparent proliferation defect and are able to compose all organ systems (Henkel et al., 1996
; Yamamoto et al., 1998
). In addition, the generation of nearly normal Ets2 deficient adult mice by complementing extraembryonic cell defects indicates that Ets2 is not essential for the proliferation of other somatic cell types. Thus Ets2 activity is uniquely required for maintenance of TS cell proliferation.
FGF4 signaling is essential for TS cell maintenance, and Erk2 is an important downstream effector during the process. Ets2 is activated downstream of growth factor receptor signaling by phosphorylation of Thr-72 (Yang et al., 1996
) (Dankort et al., 2001
; Man et al., 2003
) and is essential for FGF induction of specific genes in fibroblasts (Yamamoto et al., 1998
). Several lines of evidence suggest that Ets2 may act downstream of FGF4-Erk2 signaling to mediate TS cell proliferation and self renewal. In vivo
, FGF receptor-dependent Erk phosphorylation occurs specifically within the ExE where the TS cells reside (Corson et al., 2003
), and disruption of either Erk2 or Ets2 led to a failure of EPC and ExE formation (Saba-El-Leil et al., 2003
), both direct derivatives of the TS cells. In vitro
, depriving TS cells of Erk activity (Rossant, 2001
) or Ets2 function, as shown here, had similar adverse effects on proliferation and self renewal. Furthermore, the MAPK phosphorylation mutant Ets2A72
is haploinsufficient to support placental development, and isolation of Ets2A72/A72
TS clones showed decreased efficiency. Therefore, Ets2 likely contributes to mediating Erk2 activation by FGF4 in TS cells. However, basal Ets2 activity, independent of activation through phosphorylation of Thr-72, is sufficient for placental development and TS cell isolation albeit with decreased efficiency. Thus Ets2 also acts on additional targets that are not affected by FGF4-Erk2 signaling.
The absence of apoptosis and the gene expression changes associated with differentiation in Ets2 deficient TS cells are consistent with a switch from self renewal to beginning differentiation. However, markers of terminal differentiation, such as PL-1, PL-2 and PRP, were not induced in such TS cells. Consistently, upon growth factor withdrawal, Ets2 deficient TS cells displayed sub-optimal induction of these markers. As Ets2 level increases during induced differentiation (Supplementary Fig. S3D
), it is possible that in addition to its essential role in maintaining TS cell proliferation, Ets2 also regulates the transcription of different targets during trophoblast differentiation.
Among the list of potential Ets2 targets in TS cells, indicated by the microarray and quantitative PCR analyses, are several genes previously implicated in the self renewal of TS cells including Cdx2, Eomes and Errβ. Cdx2 is a master regulator of trophoblast commitment and self renewal (Niwa et al., 2005
; Strumpf et al., 2005
). The decreased Cdx2 RNA and protein in both Ets2 deficient TS cells and colon tissues indicates a requirement for Ets2 for optimal expression of Cdx2. Cdx2 also acts as a tumor suppressor in intestine. It is noteworthy that a second Ets2 candidate target gene, Socs2, also regulates intestinal development (Michaylira et al., 2006
The physical association of Ets2 with a highly conserved Cdx2 regulatory region and Ets2 activation of reporter constructs containing a portion of the region support a direct regulatory role of Ets2 on Cdx2 transcription. However, inactivation of Ets2 still permits residual Cdx2 RNA expression in both TS cells and colon. Furthermore, during TS differentiation, Cdx2 expression is rapidly turned off, while Ets2 expression rises modestly. Thus Ets2 contributes to the expression of Cdx2 but is not singularly responsible for its expression. The self stimulation of the Cdx2 promoter by Cdx2 protein (Xu et al., 1999
) may contribute to a threshold effect of reducing Ets2 stimulation of the Cdx2 gene. Potential binding sites for Ets2, approximately 2-kb upstream of the Cdx2 transcriptional start site represent a region of conserved DNA sequence that is occupied by Oct3/4 and Sox2 in human ES cells (Boyer et al., 2005
). The binding of ES cell transcription factors such as Oct4 and Sox2 to conserved DNA regions of key regulators of developmental lineages has been suggested to maintain potential for activation during subsequent differentiation. While the distal regulatory region of Cdx2 can mediate Ets2 activation in transient transfection analysis, the detailed mechanisms by which this region may be used in maintaining, inhibiting or facilitating expression in different cell types remains to be determined.
The forced expression of Cdx2 did not rescue the selective disadvantage of Ets2 deficient TS cells, indicating that Cdx2 down regulation is not solely responsible for the defective proliferation of such TS cells. Other candidate Ets2 target genes that are important for trophoblast development, such as Eomes, Errβ and Pace4, may contribute to Ets2 dependent TS cell self renewal. The decrease of Pace4 mRNA was among the greatest upon Ets2 inactivation. Pace4 is one of two proteases in ExE that activates Nodal, a TGF-β family member that inhibits the differentiation of TS cells (Guzman-Ayala et al., 2004
). However, the function of Pace4 is shared with another protease, Furin. As Furin mRNA is not affected in TS cells by Ets2 inactivation, decreased Pace4 expression is unlikely to exclusively account for the self renewal defect. It remains to be determined whether combination of two or more of these candidate targets can rescue the Ets2 dependent defective maintenance of TS cells.