Totipotency and lineage segregation in the human embryo

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1 Molecular Human Reproduction, Vol.20, No.7 pp , 2014 Advanced Access publication on April 3, 2014 doi: /molehr/gau027 REVIEW Totipotency and lineage segregation in the human embryo C. De Paepe 1,, M. Krivega 1,, G. Cauffman 1,2, M. Geens 1, and H. Van de Velde 1,2, * 1 Research Group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussel, Belgium 2 Centre for Reproductive Medicine (CRG), Universitair Ziekenhuis Brussel (UZ Brussel), Laarbeeklaan 101, B-1090 Brussel, Belgium *Correspondence address. hilde.vandevelde@uzbrussel.be Submitted on October 18, 2013; resubmitted on March 7, 2014; accepted on March 14, 2014 abstract: During human preimplantation development the totipotent zygote divides and undergoes a number of changes that lead to the first lineage differentiation in the blastocyst displaying trophectoderm (TE) and inner cell mass (ICM) on Day 5. The TE is a differentiated epithelium needed for implantation and the ICM forms the embryo proper and serves as a source for pluripotent embryonic stem cells (ESCs). The blastocyst implants around Day 7. The second lineage differentiation occurs in the ICM after implantation resulting in specification of primitive endoderm and epiblast. Knowledge on human preimplantation development is limited due to ethical and legal restrictions on embryo research and scarcity of materials. Studies in the human are mainly descriptive and lack functional evidence. Most information on embryo development is obtained from animal models and ESC cultures and should be extrapolated with caution. This paper reviews totipotency and the molecular determinants and pathways involved in lineage segregation in the human embryo, as well as the role of embryonic genome activation, cell cycle features and epigenetic modifications. Key words: totipotency / human preimplantation embryo / human embryonic stem cells / lineage segregation / epigenetic modifications Introduction Human preimplantation development starts with the fusion of two highly differentiated cells oocyte and spermatozoon resulting in a totipotent zygote. During the first 5 days of embryogenesis, the zygote divides, changes morphologically and forms a blastocyst. The development from a single totipotent cell into a multicellular organism encompasses intermingling of the maternal and paternal chromosomes, cleavage divisions of the cells (blastomeres), embryonic genome activation (EGA), particular cell cycle characteristics and epigenetic reprogramming. The embryo undergoes compaction on Day 4 which is characterized by increased intercellular adhesion and flattening of the blastomeres. Subsequent cell divisions and cavitation on Day 5 lead to the formation of a blastocyst (first lineage segregation). It is comprised of a fluid-filled blastocoel cavity with a compact inner cell mass (ICM) surrounded by trophectoderm (TE) cells that form a cohesive onelayer epithelium. The blastocyst further expands and hatches out of the zona pellucida. Just after blastocyst implantation into the endometrium, the ICM diverges into primitive endoderm (PE, also referred to as hypoblast) and primitive ectoderm (also referred to as epiblast, EPI; second lineage segregation). Lineage studies in mice indicate that TE cells contribute to the placenta, PE cells to the yolk sac and EPI cells to the fetus and extra-embryonic mesoderm (Gardner and Johnson, 1973; Papaioannou et al., 1975; Gardner and Rossant, 1979; Gardner, 1985). Even though human and mouse embryos seem to be morphologically similar during preimplantation development, data cannot be extrapolated without caution because important differences exist. First, blastocyst formation corresponds to days post-coitus (dpc) in mice, in contrast to Day 5 after insemination in humans (Hertig et al., 1959; Brinster, 1963; Steptoe et al., 1971). Secondly, mouse blastocysts implant at dpc, whereas human embryos undergo at least one additional round of cell division before implantation occurs between Day 7 and Day 9 after insemination (Hertig et al., 1959; Finn and McLaren, 1967; Norwitz et al., 2001; Cockburn and Rossant, 2010). Thirdly, human embryos invade into the endometrium (interstitial implantation) whereas mouse embryos attach to the endometrium and are encapsulated (secondary interstitial implantation; James et al., 2012a, b). At this moment, our understanding of human preimplantation development and the underlying regulatory mechanisms of totipotency and differentiation are limited. This is due to the scarcity of human research materials and the ethical and legal restrictions regarding the use of human embryos for research purposes in many countries. Early human embryogenesis can only be studied in vitro. Moreover, functional studies are lacking and data have been extrapolated from embryonic stem cell (ESC) lines and animal models. These authors contributed equally to the work. & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oup.com

2 600 De Paepe et al. This paper aims to review the current knowledge on totipotency and differentiation in the human embryo. The molecular determinants and pathways involved in lineage segregation are discussed as well as the contribution of EGA, cell cycle features and epigenetic modifications. We also discuss some of the data reported on human embryonic stem cell (hesc) lines. Finally, we discuss data found in human embryos and hesc that have been created by somatic cell nuclear transfer (SCNT). We refer to animal studies, mainly in the mouse, to emphasize similarities and differences between the species. Totipotency At present, there are two definitions of totipotency. According to the strict definition, totipotency refers to the ability of a single cell to develop into an adult organism and generate offspring (Edwards and Beard, 1997), of course, the cell can only demonstrate this potency after transfer into a uterus. The zygote is the ultimate totipotent cell because it is able to develop all by itself into the embryo proper with all embryonic and extra-embryonic lineages (including trophoblast supporting implantation). During preimplantation development, the embryonic cells (blastomeres) progressively lose totipotency, but it is not known when and how this occurs. Totipotency is lost because the cell is either committed or too small. Cell commitment or fate refers to an irreversible developmental restriction (i.e. differentiation) of a cell. However, the blastomeres become smaller during the early cleavage divisions. Their size, which is inversely correlated with time, may restrict their potency to develop into an organism. This limitation may be evaded using a second less stringent definition of totipotency referring to the ability of a cell to contribute to all lineages in an organism (Ishiuchi and Torres-Padilla, 2013). However, this definition may be interpreted differently by scientists going down the slippery slope (Box 1). Plasticity has been used to define an intermediate state between totipotency and differentiation. Plasticity allows the cell to have a developmental preference toward a certain cell lineage, but this preference is still reversible and thus the cell is not yet committed. In animal models, totipotency according to the strict definition has been investigated by embryo splitting. Totipotency of both blastomeres at the 2-cell stage has been demonstrated in sheep (Willadsen, 1979), but not in the mouse. Some cleavage stage blastomeres are proved to be totipotent at the 2-cell stage in mice (Tarkowski, 1959), the 4-cell stage in rhesus monkey (Chan et al., 2000) and the 8-cell stage in pigs (Saito and Niemann, 1991). Bovine is the only model where it has been demonstrated that the four blastomeres of a 4-cell stage embryo can develop into four genetically identical calves, proving that the sister blastomeres are equal and totipotent (Johnson et al., 1995). Several studies in mice demonstrated that from the 4-cell stage onwards single blastomeres need carrier cells to develop further into an organism and thus they are no longer totipotent according the strict definition (Suwińska et al., 2005; Tarkowski et al., 2010). Each blastomere of the 4-cell mouse embryo was shown by tracing experiments to give rise to trophoblast and ICM (Hillman et al., 1972). Moreover, some 4- and 8-cell stage blastomeres contribute to all lineages in chimeric mice (Rossant, 1976; Kelly, 1977; Bałakier and Pedersen, 1982) and thus are totipotent according to the less stringent definition. Similarly, aggregated inner and outer blastomeres of mouse compacted embryos (16-cell stage) are able to develop into live offspring (Suwińska et al., 2008). Finally, aggregated inner cells of early mouse blastocysts (32-cell stage) Box 1: The slippery slope of totipotency. 1. Strict definition: One cell develops on its own into a fertile organism a. Zygote b. Some single early cleavage stage blastomeres 2. Less stringent definition: One cell a contributes to all lineages b in an organism c a. One cell - One blastomere - More (aggregated) blastomeres - One (or more) pluripotent stem cell(s)8 in vivo and/or in vitro W Naive W Primed b. All lineages - Cells in all tissues and organs in vivo (embryonic and extra-embryonic layers including placenta) - Cells representing all embryonic and extra-embryonic layers in vitro based on the expression of specific markers and/or functional assays c. Organism - Live birth - Blastocyst in vitro W Post-implantation with three lineages (EPI, TE and PE) W Preimplantation with two lineages (ICM and TE) (1) In vivo chimera assay (the cell(s) is (are) injected into a carrier embryo supporting its growth and development and its descendants are found in all organs and tissues including the placenta; or (2) in vivo teratoma formation to test pluripotency (obtained after injection of undifferentiated ESC into immunocompromised mice and confirming the presence of embryonic ectoderm, mesoderm and endoderm); or (3) in vitro embryoid bodies formation to test pluripotency (by formation of 3D multicellular structures formed by non-adherent cultures of differentiating ES cells and confirming the presence of embryonic ectoderm, mesoderm and endoderm); or (4) in vitro by specific lineage differentiation. 8The definition of pluripotency, in particular the capacity to differentiate into the three embryonic germ layers, becomes problematic. Depending of their origin in the embryo, primed ESC may be pluripotent (derived from the pluripotent post-implantation EPI which develops into the three embryonic germ layers in the embryo) but naïve ESC may be more than pluripotent (derived from the preimplantation ICM which develops into embryonic and extra-embryonic lineages in the embryo but not into trophoblast cells). However, since they display more potency, they may be totipotent according the less stringent criteria (Supplementary Information, File 1). Paradoxically, primed ESC lines are more potent in vitro than naïve ESC lines. develop into blastocysts with ICM and TE cells but do not implant anymore, however, according to the least stringent definition they are still totipotent. At this stage, aggregated outer cells only develop into trophoblast vesicles and thus lost totipotency. In humans, only the zygote is proved to be totipotent according to the strict definition (Fig. 1a). One may argue that the phenomenon of bichorionic biamniotic monozygotic twinning provides evidence for totipotency of the 2-cell stage blastomeres. However, it is not known how this rare event occurs and, despite decades of in vitro culture of human preimplantation embryos in IVF laboratories, the observation of two morulas within one zona pellucida has never been reported. Moreover, according to the strict definition of totipotency, the 2-cell stage blastomeres should behave as two distinct zygotes (Herranz, 2013) thus their descendant cells should not intermingle during division and compaction (Fig. 1b). A case report describing the birth of a child after the transfer of a Day 2 cryopreserved embryo, of which only one out of four cells had survived the procedure, provided evidence that at least one of the blastomeres at the 4-cell stage is totipotent (Veiga et al.,

3 Totipotency and lineage segregation in the human embryo 601 Figure 1 Totipotency in the human according to the strict definition: (a) the zygote is totipotent because it can develop into a fertile human being after implantation into the uterus; (b) the 2-cell stage blastomeres are totipotent if they develop each individually into a human being, the descendants of the blastomeres do not intermingle during development. Manipulated human embryos cannot be transferred into a uterus to test their potency, but (c) the sister blastomeres of a 4-cell stage human embryo can develop individually into blastocysts with ICM and TE cells; (d) the descendent cells of one 4-cell stage blastomere injected with a dye contribute to both ICM and TE lineages.

4 602 De Paepe et al. 1987; Van de Velde et al., 2008; Veiga, personal communication). Indirect support for totipotency at the 4-cell stage in the human was given by splitting Day 2 embryos into four sister blastomeres that all developed in vitro into blastocysts with a compact ICM and a cohesive TE monolayer (Van de Velde et al., 2008; Fig. 1c). Finally, it has been shown that if one 4-cell stage blastomere is injected with a dye, the descendent cells contribute to both ICM and TE (Mottla et al., 1995). Obviously, (some) 4-cell stage blastomeres can contribute to both lineages and thus are not committed (Fig. 1d). Recently, it has been shown that TE as well as ICM cells from a full blastocyst can develop into ICM and TE cells, indicating that they are not yet committed (De Paepe et al., 2013), display plasticity and are totipotent according to the least stringent definition. The TE cells lose this potency from expansion onwards. For legal and ethical reasons manipulated human embryos are not transferred into a uterus to test their potency and thus totipotency according to the strict definition will never be proved in the human (Alikani and Willadsen, 2002; Van de Velde et al., 2008; De Paepe et al., 2013). In summary, the potency of the human cleavage stage blastomeres remains largely unknown, but, at least, one of the 4-cell stage blastomeres is totipotent according to the strict definition. Human blastomeres are uncommitted until the full blastocyst stage and, according to the least stringent definition, full blastocysts ICM and TE cells are totipotent. Human embryonic stem cells ESC are pluripotent cell lines usually derived from the ICM of the blastocyst and considered as a model to study embryogenesis. They can be propagated indefinitely in culture in an undifferentiated state (which is obviously not a characteristic of the zygote or blastomeres). Pluripotency refers to the capacity of a cell to develop into cells from the three germ layers in vitro and in vivo. The transcription factors POU5F1 (formerly called OCT4), SOX2 and NANOG play a major role in sustaining the undifferentiated state (Boyer et al., 2005, 2006). In mice, ESC, trophoblast stem cells (TS) and extra-embryonic endoderm stem cells (XEN) have been derived from the blastocyst (Yamanaka et al., 2006). MESC and mts lines have also been derived from single 8-cell stage blastomeres (Chung et al., 2006). The stem cell lines exclusively contribute to their progenitor lineage in chimeric animals (Yamanaka et al., 2006). ESC lines have been studied primarily in the mouse. It is clear now that there are at least two ground states of mesc (Supplementary data, File 1 and 2): (i) naïve mesc which correspond to ICM cells from preimplantation blastocysts and depend upon LIF and BMP4; and (ii) primed mepisc which correspond to post-implantation EPI cells and depend upon FGF and Activin A. In the human, pluripotent hesc lines (Thomson et al., 1998; Reubinoff et al., 2000) have been derived from preimplantation blastocysts and characterized by cell surface markers, differentiation capacity, transcriptomics and (epi)-genomics. The cultures show a high degree of heterogeneity that is partly due to variations in derivation and culture conditions (Enver et al., 2005; Osafune et al., 2008; Hough et al., 2009; Pera and Tam, 2010; Nguyen et al., 2013) and/or genetic background (Chen et al., 2009). HESC are able to differentiate in vitro into extraembryonic endoderm cells (Thomson et al., 1998; Lee et al., 2013) and trophoblast cells (Thomson et al., 1998; Xu et al., 2002; Gerami- Naini et al., 2004; Harun et al., 2006). HESC lines differentiating into trophoblast cells express specific transcriptions factors (e.g. CDX2 and GATA3), genes associated with the cytoskeleton (e.g. KRT7 and KRT8) and the extracellular matrix (e.g. COLA4), genes involved in invasion (e.g. IGF2, CDH1 or E-cadherin) and hormones (e.g. b-hcg)(marchand et al., 2011). Initially, hesc were derived from ICM cells (Thomson et al., 1998; Reubinoff et al., 2000) with the highest derivation rate from Day 6 blastocysts (Chen et al., 2009). HESC have also been derived from single 4- and 8-cell stage blastomeres (Klimanskaya et al., 2006, 2007; Feki et al., 2008; Geens et al., 2009; Ilic et al., 2009), indicating that these early blastomeres are at least pluripotent. ICM-derived and blastomere-derived hesc have similar transcriptional profiles suggesting that during in vitro culture and derivation the cells that give rise to hesc have a similar precursor cell in the embryo (Giritharan et al., 2011; Galan et al., 2013). The origin of hesc in the human embryo is unclear. Although they are generally derived from the ICM, hesc are not the counterpart of ICM cells because they do not have the same transcriptional profile (Reijo Pera et al., 2009). HESC and ICM of the full blastocyst both express surface membrane HLA-G molecules (Verloes et al., 2011), but it is not the case for all hesc lines (Drukker et al., 2006). HLA-G expression can be induced in vitro by specific culture conditions such as low oxygen (Das et al., 2007). Recently, it was shown that isolated and plated human ICM cells first develop in vitro further toward a post-icm intermediate stage and subsequently grow out into a hesc colony (O Leary et al., 2012). Based on the expression of the early germ cell markers DAZL and STELLAR it was suggested that hesc are the equivalent of early germ cells (Zwaka and Thomson, 2005). However, this hypothesis is doubtful because hesc do not easily differentiate into germ cells (Geijsen et al., 2004; Nayernia et al., 2006; Aflatoonian et al., 2009). HESC differ morphologically and functionally from mesc (Ginis et al., 2004; Schnerch et al., 2010). HESC grow as flat colonies, their undifferentiated state is maintained by adding FGF2 and/or Activin A to the culture medium. These are similar culture conditions as required for the derivation and propagation of mts lines (Yamanaka et al., 2006) and mepisc lines (Brons et al., 2007; Tesar et al., 2007). The derivation of stable human XEN and TS lines has not yet been reported either after single blastomere plating or after blastocyst plating (Douglas et al., 2009). The latter may be correlated with the fast differentiation of trophoblast into syncytiotrophoblast cells (Rossant, 2008). TS lines have been derived from rhesus monkey blastocysts but they tend to differentiate into syncytial-like cells during long-term culture (Vandevoort et al., 2007). It is now generally accepted that hesc and induced pluripotent stem cells (ipscs) obtained after somatic cell reprogramming (genetically modified by introducing transcription factors Pou5F1, Sox2, Klf4 and c-myc; Yamanaka et al., 2006) resemble more mepisc than mesc. Therefore, it has been suggested that hesc may originate from progenitor EPI cells and thus represent primed hesc. This may also explain their heterogeneity (Osafune et al., 2008; Pera and Tam, 2010; Nguyen et al., 2013) which has also been described in mepisc derived from the heterogeneous EPI (Brons et al., 2007; Tesar et al., 2007). Moreover, upon exposure to BMP4 hesc and mepisc both differentiate into PE and trophoblast cells (Xu et al., 2002; Brons et al., 2007). It is a mystery why hesc and mepisc have more in vitro differentiation capacity than mesc. For ethical reasons, it is not possible to derive ESC lines from human post-implantation embryos. Culturing hesc lines with LIF and 2i turns them into a more naïve state but these naïve hesc cannot be propagated in the long-term and differentiate (Hanna et al., 2010). Very recently, however, hesc have been stably converted into a naïve state without

5 Totipotency and lineage segregation in the human embryo 603 genetic modification by using medium supplemented with a cocktail of chemical inhibitors (Gafni et al., 2013). This supplemented medium has also been successfully used to derive stable naïve pluripotent stem cell lines from human preimplantation blastocysts. In summary, hesc represent a model to study embryogenesis in vitro. However, undifferentiated embryonic cells are only transiently present in the embryo. Moreover, hesc lines have been adapted to long-term in vitro culture conditions and may even be an in vitro artifact. Finally, hesc rather resemble mepisc than mesc, it has been suggested that hesc represent a primed stem cell state derived from post-implantation EPI cells that arise in culture after explanting the preimplantation blastocyst. Recently, a more naïve state of hesc has been obtained using specific culture conditions indicating that, similar to the mouse, there are distinct states of hesc. Therefore, data from ESC cultures should be extrapolated to the human embryo with caution. Lineage segregation Lineage segregation into TE, PE and EPI is mainly controlled by transcription factors. In mouse embryos, the first segregation is the consequence of reciprocal inhibition of POU5F1 and CDX2 in ICM and TE (Niwa et al., 2005; Ralston and Rossant, 2005; Strumpf et al., 2005). The second segregation resulting in PE and EPI is the result of mutual interaction between NANOG and GATA6 (Chazaud et al., 2006). Several models may explain the segregation of the lineages in the mouse embryo (Box 2): sorting (Chazaud et al., 2006; Dietrich and Hiiragi, 2007); position (Tarkowski and Wróblewska, 1967); polarization (Johnson and McConnell, 2004); waves of division (Bruce and Zernicka-Goetz, 2010). In mice, some of the key regulatory pathways involved in lineage segregation have been identified: Hippo signaling (Nishioka et al., 2008) and BMP4 (Home et al., 2012) in the first differentiation (Box 3) and FGF/Grb2 (Chazaud et al., 2006) in the second differentiation (Box 4). The interaction between POU5F1, NANOG and GATA6 has also been thoroughly investigated in mesc (Niwa et al., 2000; Boyer et al., 2006; Nishiyama et al., 2009) and hesc (Hay et al., 2004; Chew et al., 2005; Hyslop et al., 2005; Zaehres et al., 2005; Darr et al., 2006; Fong et al., 2008). Interestingly, during reprogramming of differentiated murine somatic cells into ipsc (another type a pluripotent cells) by Pou5f1, Sox2, Klf4 and c-myc (Takahashi et al., 2007), Pou5f1 can be replaced by E-cadherin suggesting that Pou5f1 expression is regulated by cell cell contact via E-cadherin. Interestingly, E-cadherin is linked to the WNT signaling pathway by b-catenin (Redmer et al., 2011). The first differentiation In the human, morphological differences between cells (polarization and/or position) have not been reported before compaction (Nikas et al., 1996) which establishes onset of the first differentiation. E-cadherin molecules act as homotypic receptors and contribute to adhesion of compacting cells concentrating in the areas of blastomere blastomere contact (Alikani, 2005). At the blastocyst stage, TE cells show a strong membrane localization of E-cadherin. Gap junctions (marked by connexin CX43) between the blastomeres are already detected at the 4-cell stage, but they become more dense during development and more apparent in the TE layer when compared with ICM cells (Hardy et al., 1996). Tight junctions (marked by ZO1) and desmosomes are exclusively established between the outer cells at compaction and Box 2: Lineage segregation models in mouse embryos. (1) A stochastic model was proposed in the mouse to explain the first differentiation at the compaction stage illustrating inter-blastomere variation in the amount of master proteins NANOG, POU5F1 and CDX2 followed by a phase of positional change (sorting) depending on the global differences in gene expression (Dietrich and Hiiragi, 2007). Using time lapse video, it was shown that the blastomeres move extensively at each cleavage stage (Kurotaki et al., 2007), supporting the model of cell sorting and consistent with the highly regulative capacity of the embryo. The second differentiation occurs in a similar way. Initially, the EPI- and PE-specific transcription factors NANOG and GATA6, respectively, are expressed in a random salt-and-pepper pattern in the ICM, followed by segregation into the appropriate cell lineages (Chazaud et al., 2006). (2) The inside outside model proposes that lineage segregation is directed by the position of the cell (Tarkowski and Wróblewska, 1967): outside cells develop into TE and inside cells develop into ICM. According to this model, cells on the inside and on the outside are exposed to distinct environments and different amounts of cell contact resulting into distinct fates. (3) The cell polarity model proposes that polarization is associated with differences in transcription factor expression. At the 8-cell stage, blastomeres undergo an increase in intercellular contact (compaction) and polarize along their apical basal axis (Johnson and McConnell, 2004). Polarization is characterized by the apical localization of members of the Par complex (Par3, Par6 and apkc); (Plusa et al., 2005; Alarcon, 2010). Also Cdx2 mrna becomes polarized atthe apical cortex of polarizedcells (Jedrusiket al., 2008). During the two subsequent divisions (8 16 cells and cells), the inheritance of the polarized state is influenced by the orientation of the cleavage plane in the blastomere: symmetric (conservative) divisionsgenerate polarized outer cells, whereas asymmetric (differentiative) divisions generate polar outer cells and apolar inner cells. At the 32-cell stage, the polar cells become TE whereas the apolar cells form the ICM and differentiate into EPI and PE. The two models position and polarization may work in concert to direct cell lineage segregation. Individual blastomeres separated from 2- to 32-cell stage embryos do not show a lineage-specific pattern but rather develop a unique pattern that is similar to TE (Lorthongpanich et al., 2012). It seems that the correct patterning of lineage-specific gene expression requires positional signals and cell cell interaction. (4) Another model was proposed suggesting that the first wave of asymmetric divisions would generate most of the EPI lineage whereas the second wave would generate most of the PE lineage (Bruce and Zernicka-Goetz, 2010). Cells that are not appropriately positioned change their position, gene expression profile or die by apoptosis. become clearly apparent at blastocyst expansion to support the integrity of the TE cells (Hardy et al., 1996). KRT18 expression is present in the cytoskeleton of some outer cells in the compacting embryo and further on it is found in TE at all blastocyst stages and in ICM cells facing the cavity (Cauffman et al., 2009). HLA-G, another marker for TE lineage differentiation, is present in the membrane of inner and outer cells at compaction (Yao et al., 2005). It is also transiently present in the membrane of early ICM cells (Verloes et al., 2011), but it becomes restricted to the TE cells and ICM cells facing the cavity at the moment of hatching (Verloes et al., 2011). These observations indicate that the outer cells become polarized at compaction and already obtain epithelial features induced by the environment although they are not yet committed to the TE lineage (De Paepe et al., 2013). The TE-defining transcription factor CDX2 is only detectable in the nuclei of the outer layer from blastocyst expansion onwards (Niakan and Eggan, 2013). During a short period, at the onset of expansion, POU5F1 and CDX2 are co-localized in the nuclei of TE cells. The

6 604 De Paepe et al. Box 3: The first differentiation in mouse embryos. The establishment of ICM and TE lineages in mice begins with the up-regulation of Cdx2 in outside cells followed by down-regulation of Pou5f1, Sox2 and Nanog in the same cells. Initially, Cdx2 and Pou5f1 are co-expressed in the morula; the reciprocal repression occurs in blastocysts and in mesc (Niwa et al., 2005). Depletion of maternal and zygotic Cdx2 mrna results in delayed embryo development with increased cell cycle length and problems to initiate compaction (Jedrusiket al. 2010). Inconsistent with this observation are two studies in which it was demonstrated that Cdx2 null embryos reach the blastocyst stage and collapse around the time of implantation (Wu et al., 2010; Blij et al., 2012). The embryos still cavitate and form a distinct ICM after elimination of maternal and zygotic Pou5f1 expression (Wu et al., 2013). In maternal/zygotic knockout embryos, CDX2 is not found in ICM cells and NANOG is found in cells that are scattered apart in the ICM. Later on, NANOG and CDX2 are co-localized in some EPI nuclei. Thus, the reciprocal POU5F1/CDX2 interaction does not result into the first lineage differentiation but rather maintains the ICM fate. Pou5f1 null embryos form ICM but plating these ICM does not lead to the derivation of mesc lines and the outgrowth containing a lot of Cdx2 expressing trophoblast cells. Thus, although POU5F1 is the major regulatorof pluripotency in mesc (Boyer et al., 2006), maternal POU5F1 it is not a major regulator of pluripotency in oocytes (Wu et al., 2013). Recently the Hippo signaling pathway, which plays a role in cell contact in cultured cells (Zhao et al., 2007a), has been described in the reciprocal Cdx2/ Pou5f1 repression in the embryo (Nishioka et al., 2009). The Hippo pathway involvesthetranscription factor TEAD4 andits co-activator YAP. TEAD4 acts upstream of CDX2 and is present in the nuclei of all the cells (Nishioka et al., 2008). Cellcontact and/or position mayactivate the Hipposignaling, resulting in YAP phosphorylation and subsequent nuclear exclusion in inside cells. Without the presence of YAP in the nucleus, TEAD4 is inactive and Cdx2 expression is silenced (Cockburn and Rossant, 2010). In outside cells, Yap is not phosphorylated and localized in the nuclei where it can, in cooperation with Tead4, activate Cdx2 expression. Tead2/2 embryos fail to cavitate (Yagi et al., 2007; Nishioka et al., 2008); Cdx22/2 embryos cavitate but fail to maintain TE (Strumpf et al., 2005; Jedrusik et al., 2010) and Pou5f12/2 embryos display a defective ICM (Nichols et al., 1998). The Hippo pathway may not be the only pathway involved in the first lineage segregation in the mouse embryo. Culturing mouse embryos with BMP4 blocks their development at the compaction stage and results in the unusual co-localization of CDX2 and TEAD4 in the nuclei of inner cells (Nishioka et al., 2009; Home et al., 2012). phenomenon of co-expression has also been observed in mouse, bovine and rhesus monkey embryos (Degrelle et al., 2005; Berg et al., 2011) suggesting that the segregation of the TE and ICM markers is initiated just prior to implantation. By Day 8, POU5F1 becomes restricted to a small population in the EPI indicating that distinct populations arise within this lineage (Chen et al., 2009). At this time, CDX2 appears to be down-regulated in TE. This coincides with the time when the trophoblast cells adhere and invade into the endometrium. However, whether these data represent the true in vivo situation or result from in vitro culture conditions, in particular absence of implantation into endometrial cells, is not known. Finally, whereas transcription factor binding sites for TCFAP2 that mediate CDX2-independent repression of the pluripotency marker POU5F1 are present in the mouse, they were not found in humans and cattle, suggesting alternative mechanisms for lineage commitment in different species (Berg et al., 2011; James et al., 2012a, b). So far, the molecular mechanisms that mediate the first lineage segregation in the human remain largely unknown. The Hippo signaling pathway Box 4: The second differentiation in mouse embryos. Signaling through the fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK) pathway is the earliest event known influencing differentiation of the mouseicminto theepi and PE. This pathwayleadstothe expression of the GATA transcription factors, GATA4 and GATA6, which become restricted to the PE (Feldman et al., 1995; Arman et al., 1998; Cheng et al., 1998; Chazaud et al., 2006) and the EPI marker NANOG (Mitsui et al., 2003). The transcription factor NANOG is initially present in all cells from the morula stage onwards but it becomes down-regulated in the outer cells at the blastocyst stage. Within the ICM cells, the progenitor EPI cells express Nanog and produce FGF4 whereas the progenitor PE cells express Gata6 and the Fgf2r receptor. Laminin expression seems to play a role in this lineage segregation and remains restricted to PE cells. Initially, the progenitor EPI and PE cells are distributed in a random salt-and-pepper way, the distinct lineages segregate after sorting. Grb22/2 embryos only display EPI cells in the ICM (Chazaud et al., 2006). Nanog 2/2 blastocysts have ICM cells but they fail to generate EPI (Mitsui et al., 2003). Heterozygous Nanog +/2 blastocysts have similar numbers of ICM cells in the early blastocysts when compared with Nanog +/+ blastocysts but they have fewer ICM cells in the EPI. In Nanog +/2 blastocysts fewer ICM cells are found displaying NANOG and PE formation, which depends upon functional EPI, is delayed. Another transcription factor, SOX17, has also been described in the specification of PE cell from EPI cells within the mouse ICM (Morris et al., 2010; Niakan et al., 2010; Artus et al., 2011). might be conserved between species, but information about this pathway in the human is currently not available. The other lineage, ICM cells, has also been investigated in the human. The ICM markers POU5F1, SOX2 and NANOG were already well described in hesc (Boyer et al., 2005; Hyslop et al., 2005; Zaehres et al., 2005). They bind to the promoters of their own genes forming an interconnected auto-regulatory loop controlling pluripotency and self-renewal (Boyer et al., 2005). NANOG is only present in the nuclei of some ICM cells in the full/expanding blastocyst (Hyslop et al., 2005; Cauffman et al., 2009; Niakan and Eggan, 2013). POU5F1 is found earlier in the nuclei of inner and outer cells at compaction and in ICM and TE cells at the full blastocyst stage (Cauffman et al., 2005b; Niakan and Eggan, 2013). It is down-regulated in the outer cells in the expanded blastocyst. Interestingly, NANOG is restricted to ICM cells earlier than POU5F1 (Niakan and Eggan, 2013). SOX-2 expression starts from the 8-cell stage onwards; but its nuclear expression is not restricted to the inner cells at compaction nor to ICM cells at the full blastocyst stage. SOX2 is only down-regulated in the TE cells after expansion of the blastocyst. Another transcription factor associated with the undifferentiated state, SALL4, is expressed in the nuclei of inner and outer cells at all stages from compaction till blastocyst expansion when it becomes restricted to the ICM cells. Thus, none of the markers for the undifferentiated state can be used to identify cells allocated to the ICM until expansion (Cauffman et al., 2009). The co-localization of lineage markers such as POU5F1, SOX2, SALL4, KRT18, HLA-G and the absence of CDX2 in human TE cells displaying plasticity at the full blastocyst stage explain the ability of isolated and reaggregated TE cells to reconstitute a blastocyst with a compact ICM comprising NANOG expressing cells and a cohesive TE layer (De Paepe et al., 2013). Additionally, full blastocyst TE cells can change lineage direction when they are placed in an inner position. These data suggest that full human blastocyst TE cells are not yet committed

7 Totipotency and lineage segregation in the human embryo 605 toward the TE lineage and may thus be a potential source of hesc. This potency is lost during expansion since isolated and reaggregated TE cells at this stage do not recompact anymore. This coincides with the onset of CDX2 expression (Niakan and Eggan, 2013) and the up-regulation of ZO-1 (Hardy et al., 1996) supporting the establishment of an integer and functional TE monolayer. Commitment occurs at the early blastocyst stage in mice (Suwińska et al., 2008) and in the expanded blastocyst stage in cattle (Berg et al., 2011) corresponding with the reciprocal localization of CDX2 and POU5F1. Mouse early blastocyst inner cells (Suwińska et al., 2008) and human full blastocyst ICM cells (De Paepe et al., 2013) have been shown to be capable of generating TE cells. The ability of a mouse ICM cells to differentiate into TE is controversial. Recently, it has been described that isolated mouse ICM is not able to differentiate into trophectoderm (Szczepanska et al., 2011); however, in these experiments, more advanced blastocysts were used in combination with distinct experimental procedures. Using hesc as a model to study embryogenesis in vitro, it has been found that they have the ability to spontaneously differentiate into trophoblast cells (Thomson et al., 1998; Gerami-Naini et al., 2004; Harun et al., 2006). Long-term culture of hesc lines in an undifferentiated state depends upon WNT, FGF and TGFb pathways (Box 5). The role of WNT is unclear, most likely it enhances proliferation (Dravid et al., 2005) but it has also been correlated with differentiation (Sokol, 2011). FGF and Activin A are required for the self-renewal of hesc (Amit et al., 2000; James et al., 2005; Lu et al., 2006; Xiao et al., 2006). Activin A is a member of the TGFb superfamily. BMP4, another member of the TGFb superfamily, antagonizes with Activin A and induces differentiation toward the TE lineage (Xu et al., 2002, 2008; Wu et al., 2008). Activin A and BMP4 have distinct receptors. Receptor binding transduces signals through R-SMAD proteins SMAD2/3 and SMAD1/5/8, respectively. The phosphorylated R-SMADs bind to the Box 5: Signaling pathways associated with pluripotency and differentiation in human embryos and hesc. (1) FGF: Binding of FGF to FGF receptor homodimers leads to MAPK signaling which activates transcription factors in the nucleus (Stephenson et al., 2012). (2) TGFb superfamily: Binding of homodimers of BMP4, Activin A/Nodal or TGFb to heterodimers of the Type I and Type II TGFb receptors leads to phosphorylation of cytoplasmic SMADS. The phosphorylated R-SMADs bind to the common SMAD (cosmad4) forming a complex that acts as a transcription factor for distinct target genes. Next to SMAD signaling, other non-smad pathways can be initiated by TGFb receptor activation, including MAPK. For example, TGFbII can phosphorylate PAR6 resulting in the dissemblance of tight junctions and epithelial to mesenchymal transition (Moustakas and Heldin, 2009). (3) WNT: Upon activation of the canonical WNT pathway, the b-catenin regulatory complex (Axin, APC and GSK3) is degraded. b-catenin, an E-cadherin adaptor protein which is normally degraded through phosphorylation by its regulatory complex, is accumulated in the cytoplasm and translocated into the nucleus where it will act as a transcriptional co-activator (Sokol, 2011). There is no doubt that these pathways interact with each other, e.g. GSK3 plays a key role in WNT signaling but also interferes with SMAD signaling; MAPK which plays a role in FGF signaling and the non-smad TGFb signaling pathways also has an effect on the SMAD TGFb pathway (Sakaki-Yumoto et al., 2013a). common SMAD4 and together they form a complex. This complex enters the nucleus where it directly activates transcription of distinct target genes. It has been suggested that Activin A and BMP4 may antagonize and play a role in the balance between pluripotency and differentiation by competing for SMAD4 (Xu et al., 2008). Recently, it has been found that SMAD2 plays a major role in sustaining the self-renewal of both hesc and mepisc by binding directly to the NANOG proximal promoter leading to its up-regulation (Sakaki-Yumoto et al., 2013b). Down-regulation of SMAD2 in hesc results in BMP4 signaling activation. This leads to differentiation toward endoderm (SOX17) and trophoblast (CDX2) lineages. The reciprocal interaction between CDX2 and POU5F1 further supports differentiation. This result not only supports the hypothesis that hesc and mepisc are similar, but it also demonstrates that pluripotency-versus-lineage segregation is controlled by antagonistic Activin A-versus-BMP4 interaction (Fig. 2). Finally, in mesc NANOG binds to SMAD1 limiting BMP signaling that promotes differentiation into mesoderm (Suzuki et al., 2006). It would be interesting to know whether, in the human, NANOG also binds to SMAD1/5/8 inhibiting trophoblast differentiation. FGF2 supports Activin A-dependent self-renewal of hesc via NANOG expression, but the exact mechanism is not understood (Vallier and Pedersen, 2005; Greber et al., 2008; Greber et al., 2010). The second differentiation GATA6, GATA4 and SOX17 proteins have been identified in progenitor PE cells in human expanded blastocysts (Kuijk et al., 2012; Roode et al., 2012). At this stage, some inner cells exhibit high levels of NANOG and low levels of GATA6, whereas in other cells both markers are expressed at about the same level. This pattern is similar to the salt-and-pepper distribution described in mouse blastocyst ICMs (Chazaud et al., 2006). After hatching GATA6 and NANOG are expressed in a mutually exclusive manner indicating segregation of PE and EPI, respectively (Kuijk et al., Figure 2 TGFb signaling in hesc. Activin A and BMP4 antagonize in sustaining pluripotency in hesc: SMAD2 induces NANOG expression, down-regulation of SMAD2 results in CDX2 expression via BMP4 signaling (Sakaki-Yumoto et al. 2013b). The interaction between NANOG and SMAD1/5/8 has not yet been demonstrated in hesc (dashed line).

8 606 De Paepe et al. 2012; Roode et al., 2012). SOX17 is initially detectable in early blastocysts (Niakan and Eggan, 2013). At the expanded blastocyst stage, SOX17 is highly expressed in the nuclei of all ICM cells, whereas in hatched blastocysts SOX17 expression is restricted to the putative PE within the ICM. Limited SOX17 expression has also been described in hatched blastocysts coinciding with GATA4 expression (Roode et al., 2012). GATA6 expression is detectable in the majority of SOX17 expressing cells, except for a few SOX17-positive cells within the ICM (Niakan and Eggan, 2013). In contrast to the mouse, human blastocysts express laminin in TE cells and not in PE cells, suggesting that PE lineage specification may be distinct between these two species. Finally, the epithelium markers HLA-G (Verloes et al., 2011) and KRT-18 (Cauffman et al., 2009) are present in the ICM cells facing the cavity of full and expanded blastocysts. This may be induced by the environment (blastocoel fluid) but it does not fit into the model of salt-and-pepper distribution of progenitor PE and EPI cells followed by sorting into the two distinct lineage layers. FGF/MAPK signaling plays a major role in the second lineage differentiation in mice and bovine. Bovine embryos cultured with FGF4 and heparin develop into blastocysts with an ICM that is entirely composed of PE cells (Kuijk et al., 2012). However, MAPK signaling inhibitors do not fully ablate the PE progenitor cells in bovine embryos implying other signaling pathways for second lineage segregation. In the mouse, however, pharmacological inhibition of MAPK signaling or FGF receptor inhibition in mouse embryos blocks the appearance of PE cells (Nichols et al., 2009; Yamanaka and Ralston, 2010). Mouse embryos cultured in 2i conditions exclusively give rise to the EPI lineage in the ICM. On the other hand, co-culture with FGF4 solely induces the PE lineage in the ICM (Nichols et al., 2009). Studies on human blastocysts have demonstrated that FGF/MAPK signaling is not an evolutionary conserved mechanism for the specification of EPI and PE lineages. The 2i conditions have no effect on the EPI cells in the human embryo (Roode et al., 2012). This has been confirmed by inhibiting MAPK signaling (Kuijk et al., 2012; Roode et al., 2012), indicating that FGF/MAPK signaling is not imperative for this lineage segregation in the human. The mechanism of PE lineage specification in the human remains unknown. In summary, reports on lineage differentiation in the human embryo mostly describe the expression of specific markers known from animal models and hesc. The models described in lineage segregation in mice have not been validated in human embryos. Very few functional studies have been reported. A number of studies in hesc point out distinct signaling pathways which play a role in sustaining the pluripotent state, but these pathways have not been investigated in the human embryo. Although the data in the human are limited, they are of great value because they indicate differences between distinct species and provide new insights into lineage segregation during early human embryogenesis. Embryonic genome activation Embryonic gene expression does not start immediately after fertilization. First, during early mitotic divisions, maternal mrna and proteins are degraded. Secondly, the newly formed embryo has to activate its genome, i.e. to start up a transcriptional and translational machinery to support its own growth and development. It is likely that the mrnas and proteins required for oocyte maturation and fertilization, which are dispensable for further embryo development, are degraded fast. Those that are preserved until this point are necessary to sustain the first mitotic divisions and to activate the embryonic genome and, therefore, they are only degraded after EGA. EGA is one of the mostimportant events in embryogenesis, but how exactly it is triggered is not yet completely understood. The cytoplasmic content changes dramatically during the first cleavage divisions by maternal mrna degradation and EGA. These changes may have an effect on the totipotency of the cleavage stage blastomeres. The timing of EGA onset differs between distinct species. In cats EGA starts at the 2-cell stage (Waurich et al., 2010), in sheep and cattle it starts at the 8-cell stage (Crosby et al., 1988; Kues et al., 2008) and in rabbits it starts around the first differentiation in the blastocyst (Léandri et al., 2009). In mice, clearance of RNA starts shortly after fertilization. The major part of the maternal transcripts is degraded in response to deadenylation while elimination of the other part requires participation of the products of the embryonic genome (Tadros and Lipshitz, 2009). EGA starts between the 1- and 2-cell stages followed by a peak activity between the 2- and 4-cell stages (Aoki et al., 1997; Hamatani et al., 2004; Wang et al., 2004). A relationship between the undifferentiated state and EGA has been shown for POU5F1 that appears to be critical for the expression of regulatory genes involved in transcription, translation, RNA polyadenylation and RNA degradation and thus can act as an upstream regulator of EGA in mice (Foygel et al., 2008). In the human embryo, the majority of maternal mrna is degraded between the 2- and 4-cell stage (Dobson et al., 2004; Wong et al., 2010; Vassena et al., 2011), followed by a gradual disappearance of the remaining transcripts over time (Vassena et al., 2011). Three successive waves of transcription have been found during the cleavage stages. EGA starts at the 2-cell stage with a minor wave of transcription that correspond with factors involved in transcription, protein synthesis and metabolism (Vassena et al., 2011). The second minor wave follows at the 4-cell stage. The third and major wave of transcription occurs at the 8-cell stage (Braude et al., 1988; Vassena et al., 2011) and coincides with the expression of genes involved in mrna and protein metabolism, development and differentiation. Later on, at the blastocyst stage, another major wave of specific gene expression starts involving genes that regulate further embryo development and organogenesis, implantation and placentation (Zhang et al., 2009a; Wong et al., 2010; Vassena et al., 2011). A subset of transcripts also consists of genes that are stably maintained throughout preimplantation development, e.g. housekeeping genes. Multiple waves of EGA may affect the balance between totipotency and differentiation. Several groups analyzed the temporal and spatial localization of the lineage-defining transcription factors during human preimplantation development (Cauffman et al., 2005b, 2009; Niakan and Eggan, 2013). It is clear that the totipotent human zygote does not display any nuclear expression of the three key transcription factors (SOX2, POU5F1 and NANOG) sustaining the undifferentiated state in hesc (Cauffman et al., 2009). The low cytoplasmic staining of SOX2 and POU5F1 in early cleavage stages can most likely be attributed to proteins present from the maternal stock. However, it cannot be excluded that the assays were not sensitive enough to detect nuclear localization of the proteins. Another explanation could be that the antibodies (or primers in case of mrna) are directed against a specific isoform. For instance, in the case of POU5F1_iA and POU5F1F_iB (isoforms formerly called OCT4A and OCT4B), only POU5F1_iA is associated with the undifferentiated state in human embryos and hesc (Cauffman et al., 2006). And finally, one should keep in mind that mrna expression precedes protein synthesis and transport. Thus, not the presence of the