Totipotency and lineage segregation in the human embryo.

Mol. Hum. Reprod. Advance Access published April 3, 2014

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Totipotency and lineage segregation in the human embryo

C De Paepe1,§, M Krivega1,§, G Cauffman1,2, M Geens1, H Van de Velde1,2,* 1

Research group Reproduction and Genetics (REGE), Vrije Universiteit Brussel

(VUB), Laarbeeklaan 101, B-1090 Brussel, Belgium 2

Centre for Reproductive Medicine (CRG), Universitair Ziekenhuis Brussel (UZ

*

Corresponding author: [email protected]

§

Equally contributed to the work

© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]

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Brussel), Laarbeeklaan 101, B-1090 Brussel, Belgium

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Abstract During human pre-implantation development the totipotent zygote divides and undergoes a number of changes that lead to the first lineage differentiation in the blastocyst displaying trophectoderm and inner cell mass on day 5. The trophectoderm is a differentiated epithelium needed for implantation and the inner cell mass (ICM) forms the embryo proper and serves as a source for pluripotent

differentiation occurs in the ICM after implantation resulting in specification of primitive endoderm and epiblast. Knowledge on human pre-implantation 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 embryonic stem cell 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.

Keywords: Totipotency, human pre-implantation embryo, human embryonic stem cells, lineage segregation, epigenetic modifications


embryonic stem cells. The blastocyst implants around day 7. The second lineage

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Introduction Human pre-implantation development starts with the fusion of two highly differentiated cells -oocyte and spermatozoon- resulting in a totipotent zygote. During the first five 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,

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 one layer 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 extraembryonic mesoderm (Gardner, 1985, Gardner and Johnson, 1973, Gardner and Rossant, 1979, Papaioannou et al. , 1975).

Even though human and mouse embryos seem to be morphologically similar during pre-implantation development, data cannot be extrapolated without caution because important differences exist. First, blastocyst formation corresponds to 3-3,5 days post-coitus (dpc) in mice, in contrast to day 5 after insemination in humans


cleavage divisions of the cells (blastomeres), embryonic genome activation (EGA),

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(BRINSTER, 1963, HERTIG et al. , 1959, Steptoe et al. , 1971). Second, mouse blastocysts implant at approximately 4-4,5 dpc, whereas human embryos undergo at least one additional round of cell division before implantation occurs between day 7 and day 9 after insemination (Cockburn and Rossant, 2010, Finn and McLaren, 1967, HERTIG et al., 1959, Norwitz et al. , 2001). And third, human embryos invade into the endometrium (interstitial implantation) whereas mouse embryos attach to the

, 2012a, b).

At this moment, our understanding of human pre-implantation 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.

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.


endometrium and are encapsulated (secondary interstitial implantation) (James et al.

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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

embryonic and extraembryonic lineages (including trophoblast supporting implantation). During pre-implantation 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 towards 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


totipotent cell because it is able to develop all by itself into the embryo proper with all

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demonstrated in sheep (Willadsen, 1979), but not in the mouse. Some cleavage stage blastomeres are proven 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

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 (Bałakier and Pedersen, 1982, Kelly, 1977, Rossant, 1976) 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) 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 proven to be totipotent according to the strict definition (Figure1a). One may argue that the phenomenon of bichorionic biamniotic monozygotic twinning provides evidence for totipotency of the two-cell stage blastomeres. However, it is not known how this rare event occurs and, despite


and totipotent (Johnson et al. , 1995). Several studies in mice demonstrated that

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decades of in vitro culture of human pre-implantation 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 (Figure 1b). A case report describing the birth of a child after the transfer of a day 2

procedure, provided evidence that at least one of the blastomeres at the 4-cell stage is totipotent (Van de Velde et al. , 2008, Veiga et al. , 1987) (personal communication Veiga). 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) (Figure 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 (Figure 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 proven in the human (Alikani and Willadsen, 2002, De Paepe et al., 2013, Van de Velde et al., 2008).


cryopreserved embryo, of which only one out of four cells had survived the

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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.

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, Boyer et al. , 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 information File 1 2): (1) naïve mESC which correspond to ICM cells from pre-implantation blastocysts and depend upon LIF and BMP4; and (2) primed mEpiSC which correspond to post-implantation EPI cells and depend upon FGF and Activin A.


Human embryonic stem cells

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In the human, pluripotent hESC lines (Reubinoff et al. , 2000, Thomson et al. , 1998) have been derived from pre-implantation 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, Hough et al. , 2009, Nguyen et

(Chen et al. , 2009). HESC are able to differentiate in vitro into extra-embryonic endoderm cells (Lee et al. , 2013, Thomson et al., 1998) and trophoblast cells (Gerami-Naini et al. , 2004, Harun et al. , 2006, Thomson et al., 1998, Xu et al. , 2002). 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. -hCG) (Marchand et al. , 2011). Initially hESC were derived from ICM cells (Reubinoff et al., 2000, Thomson et al., 1998) 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 (Feki et al. , 2008, Geens et al. , 2009, Ilic et al. , 2009, Klimanskaya et al. , 2006, 2007), 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 (Galan et al. , 2013, Giritharan et al. , 2011). 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


al. , 2013, Osafune et al. , 2008, Pera and Tam, 2010) and/or genetic background

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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 towards a post-ICM intermediate (PICMI) stage and subsequently grow out into a hESC colony (O'Leary et al. , 2012). Based on the

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 (Aflatoonian et al. , 2009, Geijsen et al. , 2004, Nayernia et al. , 2006). 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 (iPSC) obtained after somatic cell reprogramming (genetically modified by introducing transcription factors Pou5F1, Sox2, Klf4 and cMyc) (Yamanaka et al., 2006) resemble more mEpiSC than mESC. Therefore it has been suggested that hESC may originate from progenitor EPI cells and thus


expression of the early germ cell markers DAZL and STELLAR it was suggested that

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represent primed hESC. This may also explain their heterogeneity (Nguyen et al., 2013, Osafune et al., 2008, Pera and Tam, 2010) 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 (Brons et al., 2007, Xu et al., 2002). It is a mystery why hESC and mEpiSC have more in vitro differentiation capacity than mESC.

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 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 pre-implantation 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 postimplantation EPI cells that arise in culture after explanting the pre-implantation 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.


For ethical reasons it is not possible to derive ESC lines from human post-

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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

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 (Boyer et al., 2006, Nishiyama et al. , 2009, Niwa et al. , 2000) and hESC (Chew et al. , 2005, Darr et al. , 2006, Fong et al. , 2008, Hay et al. , 2004, Hyslop et al. , 2005, Zaehres et al. , 2005). 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 β-catenin (Redmer et al. , 2011).


mutual interaction between NANOG and GATA6 (Chazaud et al. , 2006). Several

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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 blastomereblastomere contact (Alikani, 2005). At the blastocyst stage, TE cells show a strong

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 as 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 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.


membrane localization of E-cadherin. Gap junctions (marked by connexin CX43)

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The phenomenon of co-expression has also been observed in mouse, bovine and rhesus monkey embryos (Berg et al. , 2011, Degrelle et al. , 2005) 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 downregulated in TE. This coincides with the time when the

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 CDX2independent 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 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). They activate themselves and each other and repress developmental genes. NANOG is only present in the nuclei of some ICM cells in the full/expanding blastocyst (Cauffman et al., 2009, Hyslop et al.,


trophoblast cells adhere and invade into the endometrium. However, whether these

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2005, 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 downregulated 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 8cell stage onwards; but its nuclear expression is not restricted to the inner cells at

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 like 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 towards 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 upregulation of ZO-1 (Hardy et al., 1996) supporting the establishment of an integer


compaction nor to ICM cells at the full blastocyst stage. SOX2 is only downregulated

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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

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 (Gerami-Naini et al., 2004, Harun et al., 2006, Thomson et al., 1998). Long-term culture of hESC lines in an undifferentiated state depends upon WNT, FGF and TGFβ 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 TGF superfamily. BMP4, another member of the TGF superfamily, antagonizes with Activin A and induces differentiation towards the TE lineage (Wu et al. , 2008, Xu et al., 2002, Xu 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 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


cells. The ability of a mouse ICM cells to differentiate into TE is controversial.

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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 upregulation (Sakaki-Yumoto et al. , 2013b). Downregulation of SMAD2 in hESC results in BMP4 signaling activation. This leads to differentiation towards

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 Aversus-BMP4 interaction (Figure 2). Finally, in mESC NANOG binds to SMAD1 limiting BMP signalling 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 (Greber et al. , 2008, Greber et al. , 2010, Vallier and Pedersen, 2005).

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


endoderm (SOX17) and trophoblast (CDX2) lineages. The reciprocal interaction

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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

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


expressing cells, except for a few SOX17-positive cells within the ICM (Niakan and

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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

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. Second, 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


unknown.

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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 most important 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

starts at the 2-cell stage (Waurich et al. , 2010), in sheep and cattle it starts at the 8cell 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 2and 4-cell stage (Dobson et al. , 2004, Vassena et al. , 2011, Wong et al. , 2010), followed by a gradual disappearance of the remaining transcripts over time (Vassena


blastomeres. The timing of EGA onset differs between distinct species. In cats EGA

21

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

blastocyst stage, another major wave of specific gene expression starts involving genes that regulate further embryo development and organogenesis, implantation and placentation (Vassena et al., 2011, Wong et al., 2010, Zhang et al. , 2009a). A subset of transcripts also consists of genes that are stably maintained throughout pre-implantation 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 pre-implantation development (Cauffman et al., 2009, Cauffman et al., 2005b, 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


in mRNA and protein metabolism, development and differentiation. Later on, at the

22

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 transcription factors’ mRNA, but the proteins in the nuclei should be correlated with differentiation.

The findings in human early cleavage stage embryos are supported by data in mice.

express these factors (Dietrich and Hiiragi, 2007). Pou5f1_ia (formerly Oct4a isoform) null female mice are fertile, strongly indicating that maternal Pou5f1 mRNA, which is present in the oocyte, is not a major regulator of totipotency (Wu et al. , 2013). Embryos lacking Sall4, another marker for the undifferentiated state, are also totipotent (Elling et al. , 2006). Thus the founder lineages can arise in the blastocyst without the transcription factors POU5F1 and SALL4. Based on data currently available, the transcription factors are only produced and become active in the embryonic nuclei after EGA. The major wave of transcription at the 8-cell stage in human pre-implantation embryos is particularly interesting for lineage segregation. In the human embryo, POU5F1 transcripts are already present at the 4-cell stage followed by SOX2 and NANOG at the 6-cell stage (Vassena et al., 2011). Initially, a mutually exclusive expression of -HCG/-LH and POU5F1 was found in cleavage stage blastomeres from day 3 human embryos suggesting early commitment to TE and ICM lineages (Hansis, 2006, Hansis et al. , 2004). Contradictory data, however, showed that single 5- to 8-cell stage blastomeres display a common gene expression pattern corresponding with both the undifferentiated (NANOG, POU5F1 and SOX2) and trophoblast (CDX2 and EOMES) phenotypes (however, we cannot confirm the expression of CDX2 and EOMES at


The blastomeres of early mouse embryos (before and immediately after EGA) do not

23

these early stages). This illustrates that the blastomeres are not immediately committed after EGA. Variability in RNA expression between embryos of the same stage and between individual blastomeres of the same embryo on day 3 should be interpreted with caution. First, embryo quality may affect the outcome of gene expression analysis. Embryos arrested at the early cleavage stages undergo EGA (Dobson et al., 2004).

biogenesis and poly(A) tail length modulation, whereas mRNA levels of housekeeping genes, hormone receptors and maternal factors are not significantly changed (Wong et al., 2010). Second, the sister blastomeres display a high grade of heterogeneity, partially because of the different timing of major wave of EGA onset in each of them, and autonomous development (Cauffman et al. , 2005a, Edwards and Hansis, 2005, Hansis et al., 2004, Wong et al., 2010).

In summary, the kinetics of mRNA degradation and EGA correlate with a number of intriguing observations in the human. First, maternal transcripts are largely degraded before the major onset of EGA at the 8-cell stage. Maternal mRNA degradation and EGA alter the cytoplasmic content of the blastomeres during the first cleavage divisions and this may have an effect on the totipotency of the blastomeres. Second, before the 8-cell stage the embryo does not display any of the nuclear transcription factors that have been associated with the undifferentiated state. Finally, lineage segregation does not start immediately after the first major wave of EGA.


However, they show severe repression of genes involved in cytokinesis, micro-RNA

24

Cell cycle characteristics Besides multiple signaling cascades, also the cell cycle (in general 24 hours) plays a role in the undifferentiated and differentiated states. The cell cycle (G1, S, G2 and M phases) is regulated by cyclins and cyclin dependent kinases (CDKs) (Figure 3). Cyclins are synthesised at specific stages of the cell cycle whereas CDKs are constitutively expressed. Various cyclin/CDK complexes determine progression

Interestingly, a short cell cycle (about 12 hours) has been observed in all the pluripotent cell types including ESC and early stages of embryo development (Becker et al. , 2010, Chisholm, 1988, Savatier et al. , 1994, Wong et al., 2010). In fact, a higher capacity of obtaining the pluripotent state from somatic cells has been associated with an ultrafast cell cycle (8 hours vs normal 24 hours) (Guo et al. , 2014). During the first cell cycles (cleavage divisions) the timing of the G1 phase is significantly reduced in mouse embryos (Chisholm, 1988, Moore et al. , 1996, Smith and Johnson, 1986). At the third cell cycle, the G1 phase takes only 1 hour compared to 11 hours of the normal cell cycle (Smith and Johnson, 1986). The heterogeneity in the cell cycle duration starts from the third and increases already by the fourth cell cycle (Smith and Johnson, 1986). Even during the fifth cell cycle of the cleaving mouse embryo the length of the G1 and G2 phases is about 4-5 times less than in normal somatic cells (Chisholm, 1988). The cell cycle duration of the rodent pluripotent EPI cells is significantly reduced up to 3-9 hours also due to dramatic reduction of G1 and G2 phases, and even in some cases the S phase as well (Mac Auley et al. , 1993, Stead et al. , 2002). Based on relative RNA expression analysis in mouse oocytes and 1-2-cell stage embryos, it has been shown that G1 phase-


through the distinct phases.

25

specific cyclins D1, E, CDK2 and p21 (or CDK inhibitor 1) are significantly upregulated during the second mitotic cell cycle which correlates with the onset of EGA (Moore et al., 1996). The length of the first mitotic division (1- to 2-cell stage) in mouse embryos, which is controlled by maternal factors, is associated with the capacity of the embryo to develop into blastocyst (Balbach et al. , 2012). EGA occurs at the late 2-cell stage and coincides with a longer second cell cycle (Schultz, 2002).

to the 3-cell stage (first and second cycle); indicating that the degradation of maternal transcripts is delayed or their stability is increased. On the other hand, embryonic genes are overrepresented in fast cleaving embryos pointing out that they more efficiently proceed through EGA.

Similarly, human embryos display short cell cycles during the early cleavage divisions (Wong et al., 2010). A number of cell cycle drivers, including G1 phase specific factors, are intensively activated after EGA but the check point proteins are lacking (Kiessling et al. , 2010). Due to the absence of appropriate DNA quality control at early cleavage stages, human embryos do not clear of the cells with aneuploidy, chromosome breakage or segmental aberrations. Consequently, they take part further in development and this may cause the genetic mosaicism that is observed until day 4 of human pre-implantation development (Mertzanidou et al. , 2013a, Mertzanidou et al. , 2013b, Vanneste et al. , 2009) In the human, proper cell cycle progression during the early cleavage stages determines the success of blastocyst formation as well. The combination of timelapse and gene expression studies on human IVF embryos showed a correlation between the character of the cell divisions and the transcription profile at the early


Maternal genes are overrepresented in 8-cell stage mouse embryos cleaving slowly

26

cleavage stage (Wong et al., 2010). Three cell cycle parameters have been found to predict blastocyst development: (1) the duration of the first cytokinesis, (2) the time interval between the 2- and 3-cell stage (3) and the time interval between the second and the third mitoses (i.e. synchronicity third and fourth blastomeres). Precise cell cycle progression to the 4-cell stage allows predicting embryo quality already on day 2 before the major wave of EGA. Moreover, the data indicate that embryo quality is

decrease in expression of a number of cytokine genes, while those arrested at the 4cell stage have downregulated only a few of them. This observation supports the role of the cell cycle and the importance of properly organised cleavages before the 4-cell stage in successful blastocyst formation. Finally, the precise progression to the 4-cell stage, in particular the second and the third parameter, is associated with the absence of mitotic errors (aneuploidy) at this stage (Chavez et al. , 2012). Interestingly, fragments which arise during cell division may contain micronuclei with chromosomes. These fragments may be reabsorbed by the originating cells or by neighboring cells explaining the high grade of aneuploidy in fragmented embryos. Scoring cell cycle parameters in combination with fragmentation may become the revolutionary approach needed to select the best embryo for transfer in IVF clinics.

Short cell cycles have also been observed in mESC (Savatier et al., 1994). This is correlated with the absence of certain critical regulators of cytokinesis like cyclin D and retinoblastoma proteins (Rb, prevents progression from G1 into S) (Savatier et al., 1994, Savatier et al. , 1996). Moreover, CDK2, cyclin A and E complexes are very active in mESC and lack cell cycle dependent periodicity (Stead et al., 2002).


determined before EGA. Interestingly, embryos arrested at the 2-cell stage expose a

27

Inhibiting CDK2 activity with a specific inhibitor causes the cell cycle to slow down but this is not associated with any specific cell cycle phase. In their turn, hESC expose a shortened cell cycle as well (15-16 hours) because of a truncated G1 phase (3-4 hours) and the lack of a G0 phase (Becker et al., 2010, Becker et al. , 2006). The majority of hESC are kept in the S phase. Their cell cycle is changed to normal once hESC are committed to differentiate (Becker et al., 2010,

characteristics of hESC are connected to their pluripotent capacity. A lot of data exist on the expression and function of key regulators of the cell cycle in hESC. However, most of them are contradictory and this variation is most likely caused by culture conditions and experimental design (Barta et al. , 2013). All groups agree on the presence of high levels of CDKs (CDK2, CDK4 and CDK6) and low or almost undetectable levels of CDK inhibitors (CKIs: p16, p18, p19, p20, p21, p27 and p57) (Bárta et al. , 2010, Dolezalova et al. , 2012, Egozi et al. , 2007, Neganova et al., 2009, Sengupta et al. , 2009, Zhang et al. , 2009b). CDKs are necessary for intensive proliferation and CKIs are normally upregulated in differentiating stem cells assisting the exit from the cell cycle (Dolezalova et al., 2012). Cyclins A and B are highly expressed in hESC. High levels of cyclin D2 and its partner CDK4 have been observed suggesting that those are important for G1 phase shortening, while others cyclins D1, D3 and E1 are lacking or almost undetectable in hESC (Becker et al., 2010, Becker et al., 2006). Nevertheless, the complete lack of cyclin D proteins in combination with the constitutive expression of cyclin E1 has also been reported (Filipczyk et al. , 2007). These data recall the situation in mESC where truncation of the G1 phase is achieved by constitutively high levels of CDK2/cyclin E1 allowing direct transition from M to late G1 phases (Becker et al., 2010). This explains the


Calder et al. , 2013, Neganova et al. , 2009). Therefore, the unique cell cycle

28

complete lack of most of the cyclins D necessary for the truncated G1 phase (Burdon et al. , 2002). There is an association between genes sustaining the undifferentiated state and cell cycle components in hESC. NANOG directly activates transcription of CDK6 and CDC25A and therefore stimulates the beginning of the S-phase (Zhang et al., 2009b). Downregulation of POU5F1 results in a decrease of cyclins and increase in

POU5F1 correlates with an increase in specific CDK4 and CDC25A (Greco et al. , 2007, Lee et al. , 2010). Increased cell cycle duration, in particular the G1 phase, has been associated with the onset of neural differentiation in mESC and hESC (Borghese et al. , 2010, Lange et al. , 2009), e.g. CKI p21 directly inhibits SOX2 expression in neural stem cells (Marqués-Torrejón et al. , 2013). POU5F1 and SOX2 together regulate the G1 phase specific low expression of cyclin D1 (Card et al. , 2008). Similar findings have been reported in mESC (White and Dalton, 2005).

In summary, proper cell cycle progression in early cleavage stages determines the success of human blastocyst formation. Undifferentiated cells display short cell cycles. There is a remarkable relationship between genes required for the undifferentiated state and cell cycle components in hESC. It would be interesting to know whether human blastomeres, ICM cells and hESC lines possess similar cell cycle properties.

Epigenetic modifications The identity of a cell is determined by its epigenetic state. Epigenetic changes such as DNA methylation and histone modifications influence the expression of specific


CKI p21 in human mesenchymal stem cells and hESC, while upregulation of

29

genes without altering the DNA sequence (Supplementary information File 2). The zygote, which results from the fusion of two highly differentiated gametes, is characterized by a unique epigenetic state. The parental genomes have to be reset into a totipotent state after fertilization. During pre-implantation development extensive epigenetic remodeling results in lineage segregation. Drastic epigenetic remodeling occurs during both gametogenesis and

erased and re-established and imprinted genes are set in a sex-specific way (prezygotic reprogramming). After fertilization, there is a second global wave of DNA demethylation, except for imprinted genes (post-zygotic reprogramming) (Jaenisch, 2004, Perera and Herbstman, 2011). The paternal genome, which is packaged by protamines instead of histones, is actively demethylated by factors in the cytoplasm of the oocyte. The maternal genome, on the other hand, is more resistant to the demethylating activity because it has a distinct chromatin configuration and is passively demethylated during cleavage divisions. Pre-implantation stages are characterized by DNA hypomethylation. A global wave of DNA remethylation is observed at the time of implantation.

Epigenetic events have been thoroughly investigated in mice (Supplementary information File 3). Very few data on epigenetic remodeling have been reported in the human embryo. The global level of DNA methylation has been investigated in human pre-implantation embryos obtained from in vitro matured oocytes. Some zygotes have pronuclei with a similar methylation state, whereas in other zygotes a hypomethylated paternal pronucleus is observed (Fulka et al. , 2004). The nature of this asymmetric pattern has not been investigated yet. Human embryos progressively


embryogenesis. For instance, during gametogenesis all DNA methylation marks are

30

obtain a weaker DNA methylation pattern during the cleavage stages which might be associated with the potency of the blastomeres. A global wave of remethylation occurs around the time of implantation where the TE cells shows higher DNA methylation levels than the ICM cells (Fulka et al., 2004). These data suggest that there is an epigenetic asymmetry between the two lineages, ICM and TE, in the human. DNA methylation is orchestrated by DNA methyltransferases that methylate

(DNMT1) and the de novo DNA methyltransferases (DNMT3a and DNMT3b) are expressed at almost all stages of human pre-implantation development (Huntriss et al. , 2004). Transcripts of CpG binding protein (CGBP) protecting unmethylated CpG sites from methylation were detected in ICM but not in TE cells of human blastocysts (Huntriss et al., 2004), which is consistent with the asymmetric DNA methylation pattern (Fulka et al., 2004).

X chromosome inactivation (XCI) may represent another example of asymmetry between ICM and TE cells. XCI occurs in female cells to obtain a similar dosage of Xlinked gene expression levels as in male cells and is initiated by the transcription of the long non-coding XIST RNA from the X chromosome that will subsequently be inactivated. It has been reported that female human 8-cell stage embryos display pinpoints of XIST RNA on one of the two X chromosomes (van den Berg et al. , 2009). Further in development XIST RNA is expressed in TE and PE cells from one of the X chromosomes and accumulates in a cloud on another X chromosome indicating XCI initiation. Unfortunately the status of the ICM cells was not reported in this study, while asymmetric XCI was described in mouse embryos (Okamoto and Heard, 2006). Contradictory results have been reported showing that in human


cytosine residues of CpG dinucleotides. The maintenance DNA methyltransferase

31

blastocysts XIST is expressed on both X chromosomes that remain transcriptionally active, despite XIST expression, until the time of implantation (Okamoto et al. , 2011). These opposing results may be due to technical complications and/or distinct embryo culture conditions. The XCI state of hESC is highly variable. HESC are believed to display two active X chromosomes upon derivation, but often initiate XCI due to suboptimal culture

active X chromosomes at the undifferentiated state, but randomly inactivate one of the X chromosomes upon differentiation (van den Berg et al. , 2011). HESC, however, maintain their pluripotent phenotype even in the presence of completed XCI (Bruck and Benvenisty, 2011). This supports the hypothesis that hESC resemble mEpiSCs that also display XCI and represent a later developmental stage than naïve mESC. Whereas in female human somatic cells a random XCI pattern is usually observed (Moreira de Mello et al. , 2010), the XCI pattern in TE derivatives is under discussion. A study using hESC differentiating towards the trophoblast lineage suggested that XCI in human trophoblast cells, as in mouse, is under imprinting control (Dhara and Benvenisty, 2004). Studies analysing extra-embryonic tissues and term placenta, however, reported that random XCI was common in these tissues, which may indicate that, in contrast to the mouse, XCI may not be under imprinting control in normal human placentation (Moreira de Mello et al., 2010, Zeng and Yankowitz, 2003).

Although global DNA methylation is important for the regulation of gene expression, other mechanisms are known to control gene expression as well, e.g. histone modifications. Histone acetylation is generally associated with transcriptional activity,


conditions (Lengner et al. , 2010, Silva et al. , 2008). Female mESC lines display two

32

whereas histone lysine methylation can be either activating (e.g. H3K4me3) or repressive (H3K27me3). Genes associated with H3K4 and H3K27 trimethylation have been investigated in hESC (Zhao et al. , 2007b). Three distinct categories of genes are identified: genes containing only H3K4me3 that are mainly involved in selfrenewal; bivalent genes containing both H3K4me3 (active mark) and H3K27me3 (repressive mark) involved in early development; and genes containing neither

methylation poises transcription and enables cells to flexibly modulate developmental gene expression in response to different environmental factors. The sequence of the promoter can contribute to the epigenetic mechanism that affects its regulation. In general, H3K27me3 and DNA methylation are both inversely correlated with gene expression. How they regulate pluripotency and differentiation is not fully understood. In hESC, the promoters for genes expressed late in embryo development (e.g. mesenchymal stem cells) are often CG poor and they mainly employ DNA methylation upon silencing, suggesting that DNA methylation may be required for gene repression in terminally differentiated cells (Xie et al. , 2013). On the other hand, the promoters of genes active in early embryo development (ESC, trophoblast and mesendoderm) are CG rich but they mostly engage H3K27me3 upon silencing (e.g. SOX2, Eomes, SOX17). Exceptionally, the promoter of POU5F1 and NANOG employ DNA methylation upon silencing in differentiated cells. The early embryo developmental genes remain largely unmethylated upon differentiation. In fact, early embryo developmental genes (transcription factors, signaling pathways, homeobox) are often located in large genomic domains. These domains are devoid of DNA methylation and, therefore, called DNA methylation valleys (DMVs). Many DMV genes show a bivalent state of H3K4me3 and H3K27me3, but they mostly


modification involved e.g. in immunological events. The so-called bivalent histone

33

become monovalent in differentiated cells. The flexibility fits well with the nature of early embryonic cells and stem cells keeping developmental genes poised for activation during early differentiation. These events have not been investigated during lineage segregation in the human embryo. Interestingly, although the contribution of the spermatozoon to embryo development is thought to be limited since histones are replaced by protamines, a small fraction of

particular, H3K4me3 is enriched at promoters of e.g. paternally expressed imprinted loci and certain transcription and signalling factors involved in development. On the other hand, H3K27me3 is enriched at developmental promoters that are repressed in early embryos, including many bivalent H3K4me3/H3K27me3 promoters described in hESC. These data indicate that in sperm certain loci are poised for development depending on their histone packaging. In general, developmental promoters are DNA hypomethylated in sperm but they acquire methylation during differentiation.

There are points to take into consideration when studying epigenetic mechanisms: - It is important to distinguish between patterns crucial for lineage segregation (i.e. the pre-implantation period) and the defects that can occur during pre-implantation without implications for the initial differentiation of the embryonic cells but with consequences for the period after implantation and eventually later during life. Epigenetic imprinting disorders such as Beckwith Wiedemann disease (Maher et al. , 2003) and Angelman syndrome (Cox et al. , 2002) have been associated with ART, but the origin of these diseases is unknown. - Epigenetic changes may be induced by the study procedure itself, e.g. by manipulation (Torres-Padilla, 2008). Extrinsic factors such as serum (Thompson et al.


histone-bound DNA has been found in human sperm (Hammoud et al. , 2009). In

34

, 1995) and light (Schultz, 2007) have been shown to have an effect on development through epigenetic changes. Consequently, developmental abnormalities may be introduced by assisted reproductive technologies such as hormonal stimulation, manipulation, cryopreservation, cloning (Gebert et al. , 2009, Li et al. , 2005, Thaler et al. , 2012) and associated cell culture conditions (Dumoulin et al. , 2010, McEwen et al. , 2013, Nelissen et al. , 2012). For example, vitrification has been shown to

blastocysts (Zhao et al. , 2012). This also applies to hESC, which undergo epigenetic changes upon differentiation towards different cell types, and additionally display epigenetic variation that may be inherent to the genotype or obtained after culture adaptation (Allegrucci et al. , 2007, Bibikova et al. , 2006, Doi et al. , 2009, Lund et al. , 2012, McEwen et al., 2013, Nguyen et al., 2013), the latter being stably passed on to the differentiated cells (Nazor et al. , 2012).

In summary, there are very few data reported on epigenetic modifications observed during human pre-implantation development. Human pre-implantation embryos are characterized by a global DNA hypomethylation state which might be associated with the potency of the cells. There seems to be an epigenetic asymmetry between ICM and TE lineages, but the nature of this event is largely unknown. In the human embryo there are currently no reports on histone modifications and DNA methylation patterns playing a role in lineage segregation as described in mice. However, hESC have been studied thoroughly and the nature of the cells has been associated with bivalent histone methylation marks enabling the cells to modulate developmental gene expression in response to the environment.


have an effect on the promoter methylation of Pou5f1, Nanog and Cdx2 in mouse

35

Somatic cell nuclear transfer Oocyte quality plays a major role in reproduction, it is a prerequisite for live birth and thus for totipotency. It is reflected by genetic constitution on the one hand and cytoplasmic content supporting EGA on the other hand. The cloning efficiency is largely dependent upon the reprogramming capacity of the cytoplasm. Both the oocyte and the zygote can be used for SCNT (Lorthongpanich et al. , 2010). The

necessary to reprogram the DNA (two pronuclei after normal fertilization or somatic cell DNA after SCNT) into a totipotent state and support embryo development (postzygotic reprogramming). Differences between the oocyte and the zygote may play a role in SCNT experiments. In the mouse model, therapeutic cloning (SCNT-ESC derivation for transplantation purposes) has a much higher efficiency than reproductive cloning (live birth) (Supplementary information File 4). In humans, only therapeutic cloning is allowed. Data are limited because of ethical and legal restrictions and the scarcity of human oocytes/zygotes. To reprogram adult human cells by SCNT for patient specific hESC derivation mostly enucleated mature oocytes are used. These enucleated oocytes are artificially activated (e.g. by electropulse and/or ionomycin) (Tachibana et al. , 2013, Tachibana et al. , 2009), which in normal fertilization is done by the sperm cell completing meiosis and initiating mitosis. Transcriptome comparison between human mature and fertilized oocytes revealed that their mRNA content is similar (Dobson et al., 2004). There is already some up- and downregulation of transcripts in the zygote, but the implications of these changes for SCNT experiments in the human are unknown. In IVF/ICSI embryos, the major wave of EGA occurs at the 8-cell stage (Braude et al., 1988, Vassena et al., 2011). Human SCNT embryos often arrest at the


zygote is totipotent, but the ooplasm of a mature oocyte contains all the factors

36

4- to 8-cell stage, suggesting that the somatic donor cell nucleus cannot activate embryonic genes crucial for further development (Noggle et al. , 2011). However, this arrest is not observed using caffeine during the procedure (Tachibana et al., 2013). Caffeine, a phosphatase inhibitor, is used to avoid premature activation of the cytoplasm which may be induced by the micromanipulation and results in poor embryo development. Moreover, oocytes were stained with Hoechst and subjected to

particular the mitochondria) and resolve into the early developmental arrest of the cloned embryos. The developmental arrest has also been observed in a study using human zygotes for SCNT (Egli et al. , 2011). In this study the mitotic spindle was removed after nuclear envelope breakdown in the first mitosis, however the arrest may also have been due to other technical differences. In rhesus monkey SCNT-ESC lines have been obtained (Byrne et al. , 2007), but the live birth of cloned animals has not yet been reported. Compared to normal embryos, the ICM cells of cloned embryos maintain a high level of DNA methylation and this may disturb normal embryo development after SCNT (Yang et al. , 2007).

IPSC represent another model to study the reprogramming of somatic cells. Interestingly, a privileged somatic cell state for obtaining pluripotent capacity has been associated with an ultrafast cell cycle (Guo et al., 2014). However, we do not discuss iPSC in this paper because they are pluripotent. The data on iPSC have been reviewed recently (Liang and Zhang, 2013).

In summary, mature and fertilized human oocytes have a similar mRNA content. However, it is not clear whether their cytoplasm have similar capacities to reprogram


UV irradiation to remove the DNA which can additionally damage oocyte quality (in

37

the DNA of a human somatic cell. Cloned human embryos and hESC are useful to study EGA, lineage differentiation, cell cycle progression and epigenetic phenomena.

Conclusion Information on totipotency and cell fate during embryogenesis is mainly obtained from animal models and ESC cultures, but these data should not be simply

human, however, it does not express the proteins that have been associated with the undifferentiated state. At least one of the 4-cell stage blastomeres is totipotent. The segregation of cell lineages occurs after the major wave of EGA at the 8-cell stage but the blastomeres are not yet committed until the full blastocyst stage. Particular cell cycle characteristics prevent the cells from differentiation. Lineage segregation is characterized by transcription factors that are induced via distinct signaling pathways and epigenetic modifications (Figure 4). Studies on human embryos are mainly descriptive; performing in vitro functional studies would greatly contribute to a better understanding of the key signals (signaling pathways and epigenetic modifications) that regulate differentiation during early human pre-implantation development. This will extend the basic knowledge on early embryogenesis, reproductive biology, stem cell biology and therapeutic cloning. Moreover, it will contribute to the development of new techniques in reproductive medicine focusing on efficiency and safety, e.g. by supplementing growth factors to the embryo culture medium in order to improve embryo quality and implantation capacity, or by developing non-toxic media for the cryopreservation of gametes, embryos and ovarian and testicular tissue. It may also result in better hESC culture


extrapolated to the human embryo. The zygote is proven to be totipotent in the

38

conditions and more efficient differentiation protocols to generate cells with high therapeutic potential in regenerative medicine. There are potentially numerous good quality human embryos that could be used for research: embryos carrying a genetic disease (determined after pre-implantation genetic diagnosis) and cryopreserved embryos that become available for research after the legally determined period. We could inform patients better about the need

materials, after having fulfilled their child wish. This would allow scientists to investigate more thoroughly this field of biology in the human that still remains largely unknown.

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 cella contributes§ to all lineagesb in an organismc a. One cell -

One blastomere

-

More (aggregated) blastomeres

-

One (or more) pluripotent stem cell(s)° in vivo and/or in vitro o Naive o Primed

b. All lineages -

Cells in all tissues and organs in vivo (embryonic and extraembryonic layers including placenta)


to perform research on human embryos so that they would be willing to donate their

39

-

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

o Pre-implantation 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 three-dimensional 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.

°The 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 postimplantation EPI which develops into the three embryonic germ layers in the embryo) but naïve ESC may be more than pluripotent (derived from the pre-implantation ICM


o Post-implantation with three lineages (EPI, TE and PE)

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which develops into embryonic and extraembryonic 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.

(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


Box 2: Lineage segregation models in mouse embryos

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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) (Alarcon, 2010, Plusa et al. , 2005). Also Cdx2 mRNA becomes polarized at the apical cortex of polarized cells (Jedrusik et al. , 2008). During the two subsequent divisions (8 to 16 cells and 16 to 32 cells), the inheritance of the polarized state is influenced by the

divisions generate 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.

Box 3: The first differentiation in mouse embryos The establishment of ICM and TE lineages in mice begins with the upregulation of Cdx2 in outside cells followed by downregulation of Pou5f1, Sox2 and Nanog in the


orientation of the cleavage plane in the blastomere: symmetric (conservative)

42

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 (Jedrusik et 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

distinct ICM after elimination of maternal and zygotic Pou5f1 expression (Wu et al., 2013). In maternal /zygotic knock-out 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 regulator of 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 involves the transcription factor TEAD4 and its co-activator YAP. TEAD4 acts upstream of CDX2 and is present in the nuclei of all the cells (Nishioka et al., 2008). Cell contact and/or position may activate the Hippo signaling, 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,


implantation (Blij et al. , 2012, Wu et al. , 2010). The embryos still cavitate and form a

43

2010). In outside cells, Yap is not phosphorylated and localized in the nuclei where it can, in cooperation with Tead4, activate Cdx2 expression. Tead-/- embryos fail to cavitate (Nishioka et al., 2008, Yagi et al. , 2007); Cdx2-/- embryos cavitate but fail to maintain TE (Jedrusik et al. , 2010, Strumpf et al., 2005) and Pou5f1-/- embryos display a defective ICM (Nichols et al. , 1998). The Hippo pathway may not be the only pathway involved in the first lineage

development at the compaction stage and results in the unusual co-localization of CDX2 and TEAD4 in the nuclei of inner cells (Home et al., 2012, Nishioka et al., 2009).

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 mouse ICM into the EPI and PE. This pathway leads to the expression of the GATA transcription factors, GATA4 and GATA6, which become restricted to the PE (Arman et al. , 1998, Chazaud et al., 2006, Cheng et al. , 1998, Feldman et al. , 1995) 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 downregulated 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. Grb2 -/- embryos only display EPI cells in the ICM


segregation in the mouse embryo. Culturing mouse embryos with BMP4 blocks their

44

(Chazaud et al., 2006). Nanog-/- blastocysts have ICM cells but they fail to generate EPI (Mitsui et al., 2003). Heterozygous Nanog+/- blastocysts have similar numbers of ICM cells in the early blastocysts as compared to Nanog+/+ blastocysts but they have fewer ICM cells in the EPI. In Nanog+/- blastocysts fewer ICM cells are found displaying NANOG and PE formation, which depends upon functional EPI, is delayed.

PE cell from EPI cells within the mouse ICM (Artus et al. , 2011, Morris et al. , 2010, Niakan et al. , 2010).

Box 5: Signaling pathways associated with pluripotency and differentiation in human embryos and hESC (1) Fibroblast growth factor (FGF): Binding of FGF to FGF receptor homodimers leads to mitogen-activated protein kinase (MAPK) signaling which activates transcription factors in the nucleus (Stephenson et al. , 2012). (2) TGF superfamily: Binding of homodimers of BMP4, Activin A/Nodal or TGF to heterodimers of the Type I and Type II TGF 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 TGFreceptor activation, including MAPKFor example, TGFII 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 -catenin regulatory complex (Axin, APC and GSK3) is degraded. -Catenin, an E-cadherin adaptor


Another transcription factor, SOX17, has also been described in the specification of

45

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 TGF signaling pathways also has

Authors’ role HVDV, CDP and MK prepared the manuscript; all authors edited and approved the final version.

Funding Our research is supported by grants from the Scientific Research Foundation – Flanders (FWO-Vlaanderen) and the Research Council (OZR) of the VUB.

Conflict of interest None declared


an effect on the SMAD TGF pathway (Sakaki-Yumoto et al. , 2013a).

46

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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

injected with a dye contributeto both ICM and TE lineages. Figure 2. TGFß signaling in hESC. Activin A and BMP4 antagonize in sustaining pluripotency in hESC: SMAD2 induces NANOG expression, downregulation 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). Figure 3. Cell cycle in somatic and embryonic (stem) cells. The cell cycle of a classical proliferating cell takes about 24 hours. It consists of G1 phase (11h), S phase (8h), G2 phase (4h) and mitosis (M; 1h). The cell cycle of embryonic cells is shorter (15‐16 hours) due to the truncated G1 phase. This unique characteristic of embryonic cells, including stem cells, allows them to proliferate rapidly and avoid easy onset to differentiation (red cross). The well‐known critical regulators of the cell cycle are cyclin‐dependent kinases (CDKs) and their binding partners cyclin proteins. Cyclin/CDK complex formation induces activation of CDK and consequently cell cycle progression. Inhibitors of CDKs (CKIs) prevent formation of this complex, which prevents transition from one phase to another and, therefore, inhibits the cell cycle. There are several different Cyclin and CDK proteins, each of them specific for the


blastocysts with ICM and TE cells; and (d) the descendent cells of one 4‐cell stage blastomere

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transition of the certain cell cycle phases. Complete lack of most of the Cyclins D has been proposed to be necessary for the truncated G1 phase and, therefore, shortened cell cycle in hESC (dashed red cross). Figure 4. General overview of the gradual loss in potency and lineage segregation in the human embryo. From experiments with isolated and reaggregated blastomeres it is clear

from the totipotent zygote gradually lose totipotency and finally develop into one of the three lineages (TE, EPI or PE) in the blastocyst. The segregation of cell lineages does not occur immediately after EGA. When and how the cells get committed is linked to cell cycle features and epigenetic modifications that generate distinct transcriptional programs.


that the early blastomeres are not yet committed towards ICM or TE. The cells that descend

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