During early mammalian development, genesis of the first two cell lineages, inner cell mass (ICM) and trophectoderm (TE), is dependent upon functions of key transcription factors that are expressed in a regulated and spatially restricted fashion. In this study, we demonstrate that during early mouse development, mRNA expression of transcription factor GATA3 is induced at the 4-cell stage and is consistently present during pre-implantation embryonic development. Interestingly, at the blastocyst stage, Gata3 mRNA is selectively up-regulated within the TE lineage, and GATA3 protein is abundantly present only in the TE but not in the ICM. Using mouse trophoblast stem cells (TS cells) as a model, we found that, knockdown of GATA3 by RNA interference (RNAi) down-regulates expression of caudal-type homeobox 2 (CDX2), a key regulator of the TE lineage. Chromatin immunoprecipitation (ChIP) analyses revealed that, in TS cells, GATA3 directly regulates Cdx2 transcription from a conserved GATA motif at the intron 1 region of the Cdx2 locus. ChIP analyses with mouse blastocysts also detected GATA3 occupancy at intron 1 of the Cdx2 locus. In addition, downregulation of GATA3 in pre-implantation mouse embryos reduces Cdx2 expression and inhibits morula to blastocyst transformation. Our results indicate a novel function of GATA3, in which it is selectively expressed in TE, regulates expression of key genes in TE lineage, and is involved in morula to blastocyst transformation. Genesis of the trophectoderm (TE)2 and inner cell mass (ICM) lineages during early mouse development appears to occur in two stages (1-3). First, cells are allocated to different inside and outside positions via asymmetric divisions. Then, the cells in these different positions become specified, and they become committed to restricted developmental fates. Outside cells become committed to the TE, and inside cells become ICM. Development of ICM and TE is regulated by key transcription factors that specify TE and ICM cell fate, and CDX2 has been implicated in this process (4 -6). Multiple studies indicated the importance of CDX2 in TS cell proliferation, proper function of TE, and successful implantation of blastocyst (5-8). However, molecular mechanisms that regulate Cdx2 expression in trophoblast cell lineages are poorly understood. Two other transcription factors, eomesodermin (Eomes) and TEA domain family member 4 (TEAD4), are also implicated in TE development. Mutation studies showed that the lack of Eomes also arrests blastocyst development (9). However, Cdx2 is still expressed in Eomes mutants (5). Tead4 mutants show more severe phenotypes than Cdx2 mutants and are characterized by loss of Cdx2 expression (10, 11). However, unlike Cdx2, Tead4 expression is not restricted to the TE lineage during pre-implantation development indicating that additional regulatory mechanisms are involved for the restricted expression of Cdx2 in TE lineage.Earlier, we found that, among the six members (GATA1-6) of GATA family of transcription factors, only GATA3 is abunda...

In the preimplantation mouse embryo, TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP sequencing to define genomewide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation toward the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification.A llocation of blastomeres to outside and inside positions during preimplantation mammalian development initiates specification of the first two cell lineages, the trophectoderm (TE) and the inner cell mass (ICM) (1, 2). Successful progression of TE and ICM fate specification and proper development of the preimplantation embryo depends on differential transcriptional programs that are instigated and maintained within the outer and inner cells. Gene-KO studies in mice showed TEAD4 as the master orchestrator of the TE-specific transcriptional program (3-5). TEAD4-null embryos do not mature to the blastocyst stage and TEAD4-null blastomeres lack expression of TE-specific master regulators like CDX2, GATA3, and EOMES (3, 4). However, they maintain expression of ICM-specific factors like OCT4 and NANOG.Interestingly, TEAD4 expression is maintained both in cells of TE and ICM lineages, as well as in the TE-derived trophoblast stem cells (TSCs) and ICM-derived ES cells (ESCs) (5, 6). Thus, questions are raised as to how TEAD4 selectively orchestrates the TE/TSC-specific transcriptional program but not the ICM/ ESC-specific transcriptional program. The current model predicts that the presence vs. the absence of a TEAD4 cofactor, yesassociated protein (YAP), modulates TEAD4 function at its target genes in outer vs. inner blastomeres (6), leading to the segregation of the TE and ICM lineages. However, YAP-null mouse embryos do not show preimplantation developmental defects (7), indicating that, unlike TEAD4, YAP function is dispensable during TE and ICM fate determination. It is proposed that another YAP-related cofactor, WWTR1 (i.e., TAZ), could compensate for the absence of YAP during early development (6). However, the mode of TAZ function during TE and ICM specification is unknown. Furthermore, direct targets of TEAD4 have not been identified in the TE or in trophoblast cells. Thus, definitive experiments have not been performed to conclude that loss of cofactor function/recruitmen...

Early pregnancy loss affects ∼15% of all implantation-confirmed human conceptions. However, evolutionarily conserved molecular mechanisms that regulate self-renewal of trophoblast progenitors and their association with early pregnancy loss are poorly understood. Here, we provide evidence that transcription factor TEAD4 ensures survival of postimplantation mouse and human embryos by controlling self-renewal and stemness of trophoblast progenitors within the placenta primordium. In an early postimplantation mouse embryo, TEAD4 is selectively expressed in trophoblast stem cell–like progenitor cells (TSPCs), and loss of Tead4 in postimplantation mouse TSPCs impairs their self-renewal, leading to embryonic lethality before embryonic day 9.0, a developmental stage equivalent to the first trimester of human gestation. Both TEAD4 and its cofactor, yes-associated protein 1 (YAP1), are specifically expressed in cytotrophoblast (CTB) progenitors of a first-trimester human placenta. We also show that a subset of unexplained recurrent pregnancy losses (idiopathic RPLs) is associated with impaired TEAD4 expression in CTB progenitors. Furthermore, by establishing idiopathic RPL patient-specific human trophoblast stem cells (RPL-TSCs), we show that loss of TEAD4 is associated with defective self-renewal in RPL-TSCs and rescue of TEAD4 expression restores their self-renewal ability. Unbiased genomics studies revealed that TEAD4 directly regulates expression of key cell cycle genes in both mouse and human TSCs and establishes a conserved transcriptional program. Our findings show that TEAD4, an effector of the Hippo signaling pathway, is essential for the establishment of pregnancy in a postimplantation mammalian embryo and indicate that impairment of the Hippo signaling pathway could be a molecular cause for early human pregnancy loss.

GATA transcription factors are implicated in establishing cell fate during mammalian development. In early mammalian embryos, GATA3 is selectively expressed in the extraembryonic trophoblast lineage and regulates gene expression to promote trophoblast fate. However, trophoblast-specific GATA3 function is dispensable for early mammalian development. Here, using dual conditional knockout mice, we show that genetic redundancy of Gata3 with paralog Gata2 in trophoblast progenitors ensures the successful progression of both pre- and postimplantation mammalian development. Stage-specific gene deletion in trophoblasts reveals that loss of both GATA genes, but not either alone, leads to embryonic lethality prior to the onset of their expression within the embryo proper. Using ChIP-seq and RNA-seq analyses, we define the global targets of GATA2/GATA3 and show that they directly regulate a large number of common genes to orchestrate stem versus differentiated trophoblast fate. In trophoblast progenitors, GATA factors directly regulate BMP4, Nodal and Wnt signaling components that promote embryonic-extraembryonic signaling cross-talk, which is essential for the development of the embryo proper. Our study provides genetic evidence that impairment of trophoblast-specific GATA2/GATA3 function could lead to early pregnancy failure.

Cell fate decisions are fundamental to the development of multicellular organisms. In mammals the first cell fate decision involves segregation of the pluripotent inner cell mass and the trophectoderm, a process regulated by cell polarity proteins, HIPPO signaling and lineagespecific transcription factors such as CDX2. However, the regulatory mechanisms that operate upstream to specify the trophectoderm lineage have not been established. Here we report that transcription factor AP-2γ (TFAP2C) functions as a novel upstream regulator of Cdx2 expression and position-dependent HIPPO signaling in mice. Loss-and gain-of-function studies and promoter analysis revealed that TFAP2C binding to an intronic enhancer is required for activation of Cdx2 expression during early development. During the 8-cell to morula transition TFAP2C potentiates cell polarity to suppress HIPPO signaling in the outside blastomeres. TFAP2C depletion triggered downregulation of PARD6B, loss of apical cell polarity, disorganization of F-actin, and activation of HIPPO signaling in the outside blastomeres. Rescue experiments using Pard6b mRNA restored cell polarity but only partially corrected position-dependent HIPPO signaling, suggesting that TFAP2C negatively regulates HIPPO signaling via multiple pathways. Several genes involved in regulation of the actin cytoskeleton (including Rock1, Rock2) were downregulated in TFAP2C-depleted embryos. Inhibition of ROCK1 and ROCK2 activity during the 8-cell to morula transition phenocopied TFAP2C knockdown, triggering a loss of position-dependent HIPPO signaling and decrease in Cdx2 expression. Altogether, these results demonstrate that TFAP2C facilitates trophectoderm lineage specification by functioning as a key regulator of Cdx2 transcription, cell polarity and position-dependent HIPPO signaling.