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REPRODUCTIONREVIEW

Totipotency continuity from zygote to early blastomeres: a model under revision

Michele Boiani1, Ellen Casser1, Georg Fuellen2 and Elisabeth S Christians3 1Max Planck Institute for Molecular Biomedicine, Muenster, Germany, 2Rostock University Medical Center, Institute for Biostatistics and Informatics in Medicine and Aging Research (IBIMA), Rostock, Germany and 3Sorbonne Université, CNRS, Laboratoire de Biologie du Développement de Villefranche sur Mer (LBDV), Villefranche sur Mer, France Correspondence should be addressed to M Boiani or E S Christians; Email: [email protected] or [email protected]

Abstract

The mammalian zygote is a totipotent cell that generates all the cells of a new organism through embryonic development. However, if one asks about the totipotency of blastomeres after one or two zygotic divisions, opinions differ. As it is impossible to determine the individual developmental potency of early blastomeres in an intact embryo, experiments of blastomere isolation were conducted in various species, showing that two-cell blastomeres could give rise to a new organism when sister cells were separated. A mainstream interpretation was that each of the sister mammalian blastomeres was equally totipotent. However, reevaluation of those experiments raised some doubts about the real prevalence of cases in which this interpretation could truly be validated. We compiled experiments that tested the individual developmental potency of early mammalian blastomeres in a cell-autonomous way (i.e. excluding nuclear transfer and chimera production). We then confronted the developmental abilities with reported molecular differences between sister blastomeres. The reevaluated observations were at odds with the mainstream view: A viable two-cell embryo can already include one non-totipotent blastomere. We were, thus, led to propose a revised model for totipotency continuity based on the construction of the zygote as a mosaic, which accounts for differential inheritance of totipotency-relevant components between sister blastomeres. This takes place with no preordained mechanisms that would ensure a reproducible partition. This model, which is compatible with the body of data on regulative properties of mammalian early embryos, aims at tempering the rigid interpretation that discounted maternal constraints on totipotency. Reproduction (2019) 158 R49–R65

Introduction once separated (i.e. mosaic development; for further discussion, see Lemaire 2009). Mosaic embryos (Box 3) The single-cell embryo or zygote in metazoans created were thought to have derived from oocytes organized from the fusion of an oocyte with a spermatozoon has as a patchwork of distinct territories containing various the property to generate a new organism, i.e. produce determinants, for example, molecules and/or organelles, all the necessary cells, including the germ cell lineage, which can be equally or unequally partitioned at and – in mammals – the transient extraembryonic mitosis. Consequently, only the zygote is totipotent in annexes (i.e. placenta). This property was named organisms defined as mosaic, anticipating a connection totipotency more than 100 years ago (see below). This is between the mode of partition of such determinants and the most complete definition which could be qualified as totipotency. The situation in other organisms, including the organism’s or reproductive totipotency at the animal mammals, was thought to be different by virtue of the level compared to other definitions based on cellular or regulative property of their development (Box 3). This molecular features. The first part of the review describes property should allow each two-cell blastomere to the multiple facets of the term ‘totipotency’ (Box 1) and produce a new complete organism, as stated or implied related issues raised by its meaning (Box 2). in numerous publications (Saxen 1989, Balakier & The first zygotic division in some metazoans (e.g. Cadesky 1997, Council of Europe 2003, Chuva de Sousa nematodes) is visibly asymmetric, generating a smaller Lopes & Mummery 2004, Van de Velde et al. 2008, and a larger blastomere, while in other metazoans (e.g. Mitalipov & Wolf 2009, Li et al. 2010, Lu & Zhang 2015, ascidians), this looks like an equal division which, Chazaud & Yamanaka 2016). Moreover, rabbits, pigs and nevertheless, produces two different daughter cells or sheep were occasionally born from blastomeres isolated blastomeres, as demonstrated by the subtle distinct after the third zygotic division (eight-cell stage; Moore left–right laterality of the embryos they generate et al. 1968, Willadsen 1980, Saito & Niemann 1991).

© 2019 Society for Reproduction and Fertility https://doi.org/10.1530/REP -18-0462 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via https://rep.bioscientifica.com Downloaded from Bioscientifica.com at 09/27/2021 07:01:46PM via free access

-18-0462 R50 M Boiani and others

Box 1: How to define an organism’s reproductive totipotency? Does it imply fertility? Totipotency is defined by the ability of a single cell to generate a new complete organism, which is often also presented as a ‘fertile organism’. Does it mean that totipotency must imply fertility? In this review, we consider that totipotency requires germline formation as part of the new complete organism but does not automatically require fertility, which is a physiological function. An animal may have a germ cell lineage and still be infertile due to later events, for example, intoxication or genetic mutations, which could have affected the function of the germline and precluded the formation of functional gametes. Therefore, in our opinion, it is important to make this distinction. The following quotes capture a historical transition in the importance ascribed to fertility as proof of totipotency, which appears to be progressively reduced (from ‘all the females were fertile’ to ‘normal adult capable of reproducing’ to ‘offspring including the germ line’). 1959: ‘All the females were fertile, and each female gave birth to several litters. No abnormalities of the kind observed by [Friedrich] Seidel or elsewhere were found in the young or in the more advanced embryos’ (Tarkowski 1959). 1983: ‘Totipotency might be defined as the ability of a cell or group of cells to develop into a completely normal adult that is capable of reproducing’ (Seidel 1983). 1997: ‘Full or cellular totipotency is the ability of a cell other than an oocyte to develop into an entire offspring including the germ line’ (Edwards & Beard 1997).

Box 2: Regulative vs mosaic development Regulative and mosaic development can be defined from the point of view of either the embryo as a whole or a cell as a part of the embryo. Those definitions are based on the results given by experiments manipulating the embryo or the embryonic cell. An embryo undergoing regulative development can develop harmoniously even if a part of it has been removed. This is theoretically impossible if an embryo exhibits a mosaic development, as the different embryonic cell territories are committed to strictly defined cell fates. A cell undergoing regulative development can be transplanted to another part of the embryo and form whatever structure belongs in that area instead of the structure it would have originally formed, because that given cell is still competent to receive the different signals sent by the cells surrounding it at its new location. This is possible because the fate of the tested cells is not determined at the time of transplantation. By contrast, a cell pursuing mosaic development is determined by cytoplasmic factors contained within the cell itself. This cell will form a given structure even if it is moved to a new location and is exposed to cell–cell interactions and signals that differ from its original position. When the cell is tested by transplantation to another location, its fate is already determined.

This led to a generalization and oversimplified view of be considered as acceptable models of the blastomeres those experimental data whereby loss of totipotency embedded in intact mammalian embryos. after one zygotic division was considered implausible, In this review, after having revisited historical and and both two-cell blastomeres were considered as recent data, we call for a revised model in which equally totipotent. totipotency continuity in two-cell stage blastomere In fact, new functional and molecular data produced is influenced partially by the natural partition of with mouse embryos (Casser et al. 2017, 2018) point components/ingredients that are nonuniformly to a need to reevaluate the long-held assumption of distributed in the zygotes (mosaic construction) and equal totipotency in two-cell stage blastomeres. This partially by regulative mechanisms. As a result, a viable will be the goal of the second part of this review, which embryo can consist of totipotent and non-totipotent will reexamine the original data on the reproductive blastomeres already at the two-cell stage. totipotency of single blastomeres from various mammals (mouse, rat, cow, sheep, goat and monkey) and from The multiple facets of totipotency other organisms when relevant. We focus on those experiments that tested the developmental abilities of Totipotency is usually defined by the ability of a single early mammalian blastomeres taken in isolation in a cell to give rise to all the cells of a new organism. From cell-autonomous way (i.e. excluding nuclear transfer a teleological point of view, which defines a concept and chimera production). Isolation is indispensable: considering its purpose, it is clear that zygotes are If kept together in the same zona pellucida, the sister totipotent because the goal of the one-cell stage embryo blastomeres would compete for space, raising issues is to produce an entire new organism by cell division concerning, for example, the asynchrony and order of and differentiation. After the first zygotic division, cell division which their isolation prevents. Formally, however, a second teleological argument can be raised one could still wonder whether isolated blastomeres that conflicts with the first argument. Most mammals behave as they would in the native embryo (Gulyas are viviparous and produce few embryos in the highly 1975, Gardner 1996, Edwards & Beard 1997). This sheltered environment of the mother’s body, where the question was addressed by experiments analyzing the risk of injury to one blastomere is minimal (Edwards & cell lineage of sister blastomeres kept together and Beard 1997). Therefore, it is not apparent why totipotency distinctly labeled. Since their progeny cell contribution in mammals should last over multiple cleavage stages. at a later stage (i.e. the blastocyst stage) was equivalent So, are the first two blastomeres still both totipotent? to that obtained with separated blastomeres (Mihajlovic Strictly speaking, the answer is yes if – and only if – both et al. 2015, Casser et al. 2017), isolated blastomeres can of them can operate the same functions as the zygote

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Downloaded from Bioscientifica.com at 09/27/2021 07:01:46PM via free access Blastomeres: totipotent but different? R51 in terms of generating an entire new organism. This stage were taken as a gold standard for the totipotent corresponds to the meaning Wilhelm Roux originally stage in mice, transcripts characteristic of such embryos gave to the German term ‘Totipotenz’ (Roux 1893) later (Macfarlan et al. 2012) were considered as totipotency adopted as ‘totipotence’ in English (Morgan 1901). markers. However, expression of some marker genes is This terminology was created to describe experiments a simplistic criterion that is unlikely to satisfy a complex conducted in invertebrate or lower vertebrate species state as totipotency. To date, there is no clear molecular for which isolated blastomeres had been able to give understanding of what it takes for a blastomere to be rise to premetamorphosis stages, such as pluteus totipotent (see paragraphs titled ‘Possible mechanisms larvae or tadpoles (Willier & Oppenheimer 1964). contributing to totipotency discontinuity’ and ‘In These experiments were taken as bona fide evidence for search of molecular correlates and origins of unequal totipotency, although they demonstrated only a first level blastomere potency’). of developmental regulation. There is a second layer of As a matter of fact, the term ‘totipotency’ has branched complexity in eutherian mammals owing to viviparity; off into multiple flavors and directions (discussed in a thus, the mammalian blastomere should only be granted recent review, Condic 2014), but none of them has been with a status of totipotency if its full development able to reflect consensus. Why is defining totipotency in utero is observed, including the generation of all the so challenging? This can be partly attributed to the cell lineages (extraembryonic and embryonic tissues, following three considerations. Firstly, this property including the germ cell lineage). This facet of totipotency evolves as embryonic development progresses and can be qualified as reproductive, developmental or a single, unique and rigid definition simply might not organismal (Box 1, Fig. 1A). work. Secondly, this property must be empirically Because this definition of totipotency entails gestation tested according to chosen criteria that are used for its and full development, which in some mammalian definition (either reproductive, or cellular or molecular species are impractical or unethical to test, its content definition of totipotency, see above), and this choice was extended and ‘totipotency’ was employed in a more may vary from one study to another. Thirdly, historical permissive way, depending on the biological context. literature had associated totipotency with the concepts Totipotency, for example, was used to qualify a cell of regulative or mosaic development, but those concepts which simply contributes to ‘all’ tissues in chimeras have themselves rapidly evolved. As more recent or differentiates into ‘all’ cell types where in practice discoveries attract more attention, older knowledge can not ‘all’, but a representative set of cell types can be be forgotten even if it can still provide a useful compass. considered, specifically including both embryonic and Consequently, those words (totipotency and regulative extraembryonic cell types in mammals (Suwinska et al. and mosaic development, Fig. 1C, D and E) are diversely 2008; Fig. 1A and B). We would like to group the latter combined and can correspond to various biological ones into cellular totipotency to distinguish between realities, as we will discuss in the next paragraph. those possible definitions. In addition, the search for a molecular definition Revisiting totipotency in light of the historical and of totipotency led to an even more permissive debated concept of mosaicism vs regulation interpretation: A cell would be defined as totipotent if it expressed in high levels of certain transcripts or At the end of the 19th century, two scientists, Hans Driesch totipotent markers. Because embryos at the two-cell and Wilhelm Roux, designed experiments to address the

Figure 1 Definition of totipotency and interrelation of the concepts of totipotency with regulative and mosaic development. (A and B) Schematize the two main definitions of totipotency. (C, D and E) Schematize the interrelations. (A) A totipotent cell is an embryonic cell or blastomere able to generate a new entire organism. This also corresponds to reproductive totipotency (see text for detail). (B) A totipotent cell is a cell or embryonic cell or blastomere able to differentiate into all the different cell types existing in the organism. (C) At the first cleavage, if the two-cell blastomeres are both totipotent, they can both give rise to an entire organism (in green) by regulative development. (D) At the first cleavage, if the two-cell blastomeres are both not totipotent due to mosaic construction of the zygote, they fail to produce an entire organism after having initiated development (in blue or red). (E) The mosaic construction of the zygote may allow the transmission of all the critical determinants for totipotency and successful development into only one (green) of the two daughter cells while the other undergoes unsuccessful development (in red). The successful blastomere can undergo development by itself thanks to regulative properties. https://rep.bioscientifica.com Reproduction (2019) 158 R49–R65

Downloaded from Bioscientifica.com at 09/27/2021 07:01:46PM via free access R52 M Boiani and others problem of early embryo totipotency. They used two did not envision any link between this process and different animal species and methodologies. Driesch’s the mosaic construction of the zygote. By contrast, experiment separated the blastomeres of a European green he was convinced that regulative mechanisms could sea urchin (Echinus microtuberculatus, later renamed reformulate the developmental processes so that the Psammechinus microtuberculatus) two- or four-cell stage single isolated blastomere could produce an entire embryo and showed that those isolated blastomeres tadpole (McClendon 1910). could generate completely albeit smaller larvae, thanks The elegant simplicity of early experiments conducted to regulative development (Driesch 1964 (1892)). Two- in sea urchin and frog proved to be a challenge in and four-cell blastomeres were, therefore, considered mammals. An experimental study of mammalian as totipotent, and the successful development of those totipotency was not possible without two important blastomeres was considered as regulative (Fig. 1C). techniques: (1) Cell culture that was necessary to evaluate Unfortunately, experiments by Driesch and even more totipotency in terms of blastocyst formation (stage of the recent ones by others were embellished in the collective primary regulation with the formation of the inner cell memory: Only 5–10% of the two-cell blastomeres led mass, ICM, and the trophectoderm, TE) and biomarker to successful complete development (Cameron et al. gene expression and (2) embryo transfer into the uterus 1996). Thus, the development of monozygotic (MZ) of a foster mother that was necessary to demonstrate the twins or quadruplets was rarely observed, and when this complete formation of a new organism as required by was the case, a mix of normal and abnormal embryos the reproductive definition of totipotency. Mammalian was clearly visible. Those observations suggest that sister embryo culture became a reliable method only in blastomeres are not as equally totipotent as once thought the 1960s, thanks to the work of Whitten, McLaren, (Plough 1927, Marcus 1979). Chang, Brinster and Biggers (reviewed in Biggers 1998) Roux had chosen embryos from the green frog (Rana and embryo transfer, which was pioneered by Walter esculenta) for his experiments, and he tested the ability of Heape as early as 1890, was not reliable for many years one of the two-cell blastomeres to develop after damaging (Biggers 1991). In 1942, 50 years after Driesch and Roux, the second blastomere. The lack of complete development Nicholas and Hall were able to separate blastomeres of of the untouched blastomere was mistakenly interpreted two-cell rat embryos to obtain complete but not fully as distinct potency of each blastomere and viewed as normal embryos (Nicholas & Hall 1942). In the 1950s, evidence for mosaicism (Fig. 1D). the laboratory of Albert Dalcq and Jacques Mulnard In fact, the outcomes in Roux’s experiment were was working on the enzymatic activities characterizing influenced by the presence of the injured blastomere, the early development of rat embryos. They observed a as later results showed (McClendon 1910). diffuse phosphatase activity (diffuse reaction) which was McClendon reported in his paper how he was able unevenly located in blastomeres, leading them to think to eliminate one blastomere by aspirating its content that it could correlate with ICM-TE segregation. Their so that the remaining blastomere could develop in a findings could be interpreted as a possible molecular rather normal tadpole. He discussed the way in which explanation for the early transition from totipotency frog zygote cleaved, bisecting the gray crescent – a to early differentiation (Mulnard 1955; reviewed in less pigmented area opposite the sperm entry point – Mulnard 1986). Although those data were criticized later equally to produce ‘two totipotent halves’. Although by Solter and collaborators (Solter et al. 1973; Box 3), McClendon had already understood, with others, the the diagram of ‘segregation theory’ shown in Fig. 2A importance of the partition of the gray crescent, he is instructive, as it prefigures later studies on two-cell

Box 3: Controversies, debates and ‘forgotten’ literature Solter and collaborators summarized the current controversy existing regarding Mulnard’s work in the following terms in their 1973 paper: (studies by other authors) ‘suggest that all blastomeres up to a certain stage are equipotent and possess remarkable regulative capabilities’ (and this view is) ‘at odds with conclusion proposed by Mulnard (1955, 1965)’. In addition, ‘since specific cytoplasmic localization plays an important role in early embryonal development and differentiation’ in other species such as sea urchin (Davidson 1968), Solter and collaborators wanted to repeat the experiments carried out by Mulnard to confirm or confute cytoplasmic localizations in mammalian embryos, but they did not succeed in their attempt. Importantly, in their conclusion, they remained cautious and took into account Davidson’s point of view establishing that cytoplasmic localization and regulative properties are not mutually exclusive. They ended their debate as follows: ‘We cannot exclude the existence of cytoplasmic localizations in mammalian embryo but there is no positive evidence for it’ (Solter et al. 1973). It should be noted that the clear distinction between organisms exhibiting either regulative or mosaic development was historically transient at the end of the 19th century, followed by a reaction against the possible existence of mosaic development. As indicated above, these two modes of development are not mutually exclusive (Box 2). A key difference between mosaic and regulative developmental processes lies in the respective role of intrinsic or extrinsic molecular mechanisms controlling – and possibly restricting – cell fate and at which time during development they operate. At early stages, the orientation of the cleavage planes regarding the distribution of ooplasmic components involved in cell determination might be of paramount importance to the outcome. The views in favor of pre-mosaicism and pro-regulative nature of the early mouse embryo have led to still heated debates (Glover 2006). Recommended reading: Lawrence & Levine (2006), Hiiragi et al. (2006), Johnson et al. (2009), Kubiak et al. (2006).

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Downloaded from Bioscientifica.com at 09/27/2021 07:01:46PM via free access Blastomeres: totipotent but different? R53 diversification with the hypothesis of heterogeneously unequally to the two-cell stage blastomeres (Fig. 2B). localized egg components (Fig. 2A). Hence, Seidel suspected two-cell (or even four-cell) Still in the 1950s, Friedrich Seidel and Andrzej blastomere diversification. Unfortunately, his study Tarkowski used blastomere lethal injury (Roux’s was published in German, which limited its spread experimental approach) independently to determine across the international scientific community: Die the totipotency of early blastomeres in rabbit and Entwicklungsfähigkeiten isolierter Furchungszellen aus mouse, respectively. Rabbits could still be born when dem Ei des Kaninchens Oryctolagus cuniculus (The one of the two- or four-cell stage blastomeres was developmental abilities of isolated embryonic cells from kept alive following ablation of the other blastomere(s) the egg of the rabbit Oryctolagus cuniculus; Seidel and transplantation into a foster female (Seidel 1952, 1960). While cited in the English-written scientific 1960). Seidel’s careful analysis of the blastocysts (stage literature (Denker 1976, Gardner 1996, Edwards 2005, of primary regulation) identified three types of rabbit Suwińska 2012), Seidel’s work probably did not receive embryos: (1) The normal blastocyst with well-formed all the attention it deserved. Tarkowski investigated the ICM, (2) the blastocyst made of a double layer of ability of the mouse two-cell blastomeres to develop into epithelial-like cells and (3) the blastocyst without ICM. blastocysts and then into offspring (Tarkowski 1959). Seidel hypothesized that those three types of embryos He described in his seminal report published in Nature were obtained as a consequence of the egg organization, how this capacity is variable and, similar to Seidel, he whose components (e.g. a ‘Bildungszentrum’, the observed some blastocysts without ICM. Furthermore, German term for organization center) were allocated it can be deduced from his data on postimplantation development that some embryos lacked the ability to build a functional placenta; thus, the totipotency of the blastomere tested was not confirmed in those cases (Tarkowski 1959). Two conclusions could be drawn from those pioneer studies. Firstly, they revealed that the blastomere tested for its totipotency could not always be developmentally successful for technical or biological reasons, and this observation might have hampered any strong, definitive conclusion about its status. Secondly, several investigators (e.g. McClendon in frog, Seidel in rabbits) had envisioned the correct partition of key determinants in daughter blastomeres as critical for the maintenance of their totipotency. Hence, their work already supported the idea that the initial mosaic composition of the zygote could be followed by regulative processes that would lead to a new organism from a single blastomere (definition of organism’s/reproductive totipotency). However, the existing literature does not clearly reflect this functional association between the three words (totipotency and regulative and mosaic development), as demonstrated by Figure 2 Transmission of egg (either unfertilized or fertilized) components to the two-cell embryo. (A) Scheme adapted from Fig. 7 a rather unproductive PubMed search made on February shown in Mulnard (1986) and related studies using rat embryo. 9, 2019 (a combination of the three terms returned no Enzymatic activity (Mulnard’s diffuse reaction) identified in a items while the combined key words ‘totipotency’ cytoplasmic region of the fertilized egg is allocated to one of the and ‘regulative’ returned six items). We would like to two-cell blastomeres or partly to the two blastomeres, depending on reinvigorate this association in our current discussion the orientation of the first cleavage plane. This enzymatic activity is featuring totipotency, and regulative and mosaic ultimately inherited by the cells forming the inner cell mass (ICM) of development as three interrelated concepts which should the blastocyst and not the cells of the trophectoderm (TE). Thus, the first differentiation event during development, ICM versus TE, is be integrated together with a fourth one, the organization correlated to this enzymatic activity. (B) Schema adapted from Seidel of the egg materials to be inherited by the zygote after and related studies using rabbit embryos (Seidel 1960). Seidel’s fertilization, i.e. mosaic construction of the zygote (Fig. 2). scheme hypothesizes that components stored in the egg can be transmitted in different ways depending on the 1st cleavage plane Totipotent and non-totipotent blastomeres found in (1, 2, 3 or 3′). Developmental capability of the two-cell blastomere is illustrated by the type of blastocyst this single embryonic cell can early embryos: imperfect regulation or mosaicism? give rise to: A normal blastocyst with ICM and TE, an abnormal blastocyst lacking ICM or a didermic blastocyst made of a double Additional studies since the first experiments in layer of cells without defined ICM. The dashed line in (A) and (B) mammals have addressed the question of early labeled 1 represents the axis of the fertilized egg or zygote. blastomere totipotency. The equal totipotency of each https://rep.bioscientifica.com Reproduction (2019) 158 R49–R65

Downloaded from Bioscientifica.com at 09/27/2021 07:01:46PM via free access R54 M Boiani and others blastomere composing an early embryo has particularly remained an open issue, because its testing requires the dissociation of the embryo and an isolation of each blastomere to prove its full development (twin, quadruplet). This is a complex task because blastomeres must be separated without damaging them and, even more importantly in viviparous species, development to term must occur in utero and requires the formation of extraembryonic tissues. Therefore, from a technical point of view, the experiment requires embryo transfer in the foster mother, and from an embryologic point of view, it implies the success of a first level of regulative development or ‘primary regulation’ (from the original German term ‘Gestaltungregulation’, F. Seidel). Starting from the single isolated blastomere, the ‘primary regulation’ enables the production of embryonic and extraembryonic tissues (ICM and TE). Because it is impossible to achieve full development in the absence of this first level of regulative development, the totipotency of two-cell blastomeres can be judged based on either blastocyst formation and biomarker gene expression (bona fide totipotency) or full development (retrospective-reproductive totipotency). Therefore, we would like to focus on studies that kept track of the native pair of blastomeres and scored development in order to answer two distinct but complementary questions. The first question asks how often two-cell blastomeres of the same embryo are equally totipotent, while the second question asks how prevalent totipotency still is after the two-cell stage. Figure 3 Ability of blastomeres to develop as singlets or twins after How often are two-cell blastomeres of the same transfer of embryos derived from individual pairs of two-cell stage blastomeres (A) and ability of individual blastomeres from various embryo equally totipotent? Isolated two-cell blastomeres stages to exhibit totipotency (B). (A) Distribution of initiated exhibited a high frequency of blastocyst development pregnancies for twins created by blastomere separation at the (92%), similar to non-manipulated embryos, in our two-cell stage. Data from nine studies (1 to 9; T = total) are shown and recent study in mice (Casser et al. 2017). By contrast, number of embryos for each category is indicated in each block. in this and in another four studies, 30 ± 3.7% of the Study 1: Casser et al. (2017). Study 2: Togashi et al. (1987). Study 3: pairs of isolated two-cell blastomeres or their derivative Tsunoda and McLaren (1983). Study 4: Wang et al. (1997). Study 5: Sotomaru et al. (1998). Study 6: Hashiyada (2017). Study 7: Willadsen blastocysts were able to develop successfully into (1979). Study 8: Matsumoto et al. (1989). Study 9: Mitalipov et al. pairs of liveborn mice after transplantation into the (2002). The total number of embryos pulled from all the studies/ genital tract of foster mothers (Tsunoda & McLaren categories is shown. (B) Graph showing the percentage of blastomeres 1983, Togashi et al. 1987, Wang et al. 1997, Sotomaru exhibiting totipotency (y axis) either by achieving full development or et al. 1998, Casser et al. 2017) (Fig. 3A, studies 1–5). by generating stem cell line (*) calculated at various embryonic stages The relatively low occurrence of equal totipotency in and as a function of the percentage of the volume of the blastomere pairs of two-cell blastomeres may not be blamed on (x axis) used perform the experiments. The approximate ratio of the blastomere volume to the total embryo volume is indicated in technical issues, since a high number of embryos (over parentheses. Data summarized in (B) are extracted from the following 500 in total) were used for embryo transfer in different studies. Mouse: two-cell data from (A), four-cell and eight-cell data laboratories. As Seidel pointed out, not all blastocysts from Tarkowski and Wroblewska (1967) and from Wakayama et al. are developmentally competent (Seidel 1960), and (2007). Sheep: Willadsen (1981). Rabbit: Moore et al. (1968). this can be assessed by their cellular composition, via Pig: Saito & Niemann (1991). MZ: monozygotic pair (twins). scoring for the presence/absence of ICM or by a more sophisticated approach using molecular markers for cell able to determine that only 27% of embryos had a subpopulations (TE and epiblast or primitive endoderm presumptively functional epiblast in both members of in the ICM). These subpopulations arise from two the pair (≥4 NANOG-positive cells, Morris et al. 2012). consecutive phases of cell lineage commitment, ICM This low percentage could be a consequence of the vs TE (1st phase) and epiblast vs primitive endoderm artificial separation of the blastomeres, which could (2nd phase) (De Paepe et al. 2014). Using molecular have impinged on their adaptive/regulative abilities. In markers (Casser et al. 2017), the authors were order to exclude the effect of artificial separation, each

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Downloaded from Bioscientifica.com at 09/27/2021 07:01:46PM via free access Blastomeres: totipotent but different? R55 blastomere of an intact two-cell embryo was injected some positive data, a proportion ranging from 14.7 to with a different fluorescent dye to perform lineage 40.7% (mean = 27.7 ± 13%, n = 353) of the two-cell tracing at the blastocyst stage. Again, only a small embryos produced liveborn MZ pairs after blastomere group of embryos (22%, 8/36) exhibited a balanced isolation and transfer to foster mother (Fig. 3A). We feel contribution of both blastomeres to the formation of the that the gap between the expected 100% and the actual epiblast (Casser et al. 2017). While pursuing a distinct 27.7% should be smaller if both sister blastomeres were scientific question, another research group using a indeed totipotent, even though it is unlikely that all the similar experimental approach reported that only a small blastomeres from all embryos could achieve a normal proportion of embryos (33%, 9/25) had a concordant development (e.g. not all concepti have a normal set of ability to form epiblast cells from both blastomeres of chromosomes). Thus, the answer to the first question is the undivided two-cell stage (Mihajlovic et al. 2015). All ‘no’, not all two-cell blastomeres are equally totipotent. together, these data collected from mouse embryos led This impacts directly on the second question (how us to doubt that the two-cell blastomeres could always prevalent is blastomere totipotency after the two-cell be equally totipotent. stage?), as we know that a substantial proportion of One must be cautious before translating findings two-cell blastomeres have already lost their totipotency obtained in mouse embryos directly to other mammalian after the first zygotic cleavage. Conversely, totipotency species and establishing their general validity, because might be restrained to only one of the sister blastomeres Mus musculus development exhibits some specificities in mammalian two-cell embryos, while this possibility whose impact on totipotency is not yet clear. The duration needs to be further evaluated at later stages. of the two-cell stage, for example, is extended in mouse The second question to address is whether blastomere embryos, which later form a tridimensional egg cylinder totipotency is substantial past the two-cell stage. The with the epiblast developing inside (‘inversion’ of the ability of all the blastomeres of a single four-cell stage germ layers) as opposed to the more flat embryonic mammalian embryo to develop autonomously into disc observed in other mammalian species (Johnson & liveborn animals was documented only in two studies Alberio 2015). In addition, comparative gene expression (Willadsen 1981, Johnson et al. 1995). It, thus, appears analysis of mouse and human embryos reveals that this ability is very difficult to demonstrate when distinct patterns in the appearance and localization the embryo has more than two blastomeres, because of lineage-specific transcription factors Niakan( & all of them have to be preserved alive and undamaged. Eggan 2013). Unfortunately, studies reporting relevant Nevertheless, reproductive totipotency could be tested experiments of blastomere isolation carried out in non- successfully for some of the blastomeres at the four-cell murine mammalian species are scarce. After Seidel’s stage in cow and monkey (Johnson et al. 1995, Chan work in rabbit, only one study used this species to et al. 2000) and up to the eight-cell stage in rabbit, determine the developmental competence of isolated sheep and pig (Moore et al. 1968, Willadsen 1980, Saito blastomere up to the eight-cell stage (Moore et al. 1968). & Niemann 1991). In humans, the intentional testing The percentage of developmental success decreased of blastomere totipotency by complete development of with the embryonic stage and the authors concluded liveborn babies is precluded by ethical considerations, that ‘increasing cell stage of the parent egg might suggest as this would be assimilated to an attempt at human that every blastomere is not totipotent’. However, this cloning (Editorial 1994). Serendipitously, Veiga and conclusion was not formally demonstrated, as pairs collaborators obtained a living birth after transfer of a or quadruplets of isolated early blastomeres had not human embryo in which only one blastomere out of four been tested simultaneously (Moore et al. 1968). After had survived following freezing/thawing (Veiga et al. Dalcq’s work in rat, one study reported the complete 1987). There is no report of single blastomeres producing development of 9 out of 19 pairs of rat two-cell liveborn offspring after the eight-cell stage in mammals. blastomeres (Matsumoto et al. 1989). In cattle, three Only one case of ultrasound-detected early pregnancy out of ten twin pairs produced by two-cell blastomere from a 16-cell stage blastomere in pig could be mined in separation and culture were born using either Japanese the literature (Saito & Niemann 1991; Fig. 3B). black or Holstein cattle (Hashiyada 2017). In goat, two Even when blastomere totipotency was assessed out of five pairs created at the two-cell stage were born as at an intermediate or primary stage of regulation pairs (Tsunoda et al. 1984). In sheep, five out of ten pairs (e.g. peri-blastocyst), the rate of developmental success created at the two-cell stage and transferred were born was low. In mice, we found two reports analyzing how as MZ pairs (Willadsen 1979). In nonhuman primates, dissociated four-cell embryos developed to the peri-/early no MZ pairs but one singlet were born after separation postimplantation stage (Tarkowski & Wroblewska of four two-cell embryos (Mitalipov et al. 2002) (Fig. 3A, 1967, Rossant 1976). An average of 30% of the isolated studies 6–9). blastomeres were able to form blastocysts, while only 1 Overall, taking into account all the mammalian out of 25 could form a proper embryo after implantation species examined and all the experiments with at least (Rossant 1976). When the blastomeres were isolated

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Downloaded from Bioscientifica.com at 09/27/2021 07:01:46PM via free access R56 M Boiani and others from eight-cell stage embryo, the percentage of Possible mechanisms contributing to blastocysts decreased significantly (0% in Rossant totipotency discontinuity 1976; 11% in Tarkowski & Wroblewska 1967). More recent studies aimed to determine whether this might Several mechanisms may contribute to the emergence be due to commitment/restriction of blastomeres to a of interblastomere differences and the variation in the distinct cell lineage (Tabansky et al. 2013). Tabansky manifestation of totipotency. They can be analyzed and colleagues used a variant of the multicolor from the regulative and/or mosaic point of view (Fig. 4), Brainbow system to generate distinct color labeling of and they are not mutually exclusive. Nevertheless, each blastomere at the four-cell stage and trace their their relative importance may vary over time, progenies at the blastocyst stage (Tabansky et al. 2013). adding complexity to the developmental events we All the colors were visible in the different lineages for described above. 30% of the blastocysts developed, revealing that the Firstly, several biological processes can occur contribution of the four blastomeres to ICM or to the at variable speeds and, thereby, asynchronously entire blastocyst was homogeneous. By contrast, colors between sister blastomeres, such as (de)adenylation or in the remaining two-thirds of the blastocysts were degradation of maternal mRNAs (Fig. 4A). Since zygotic differently distributed among cells from the ICM and or embryonic genome activation (i.e. ZGA or EGA) are in the whole blastocyst. Thus, the four blastomeres in controlled partially by maternal mRNAs, it follows that this larger group did not contribute equally to the ICM ZGA-EGA can also take place asynchronously in sister lineage, and this could be explained by the fact that blastomeres. This is accompanied by a reorganization of they were not all equal in terms of cellular totipotency embryonic chromatin. It was shown, for example, that (Tabansky et al. 2013). In humans, Van de Velde and chromatin mobility is higher in two- than in eight-cell colleagues dissociated six embryos at the four-cell mouse embryos (Boskovic et al. 2014), so this parameter stage and one of them gave four blastocysts containing could also vary slightly between the two-cell stage visible ICMs, which, however, included a variable blastomeres. These observations suggest that ZGA-EGA number of NANOG-positive cells (Van de Velde et al. participates in the genesis of the first interblastomere 2008), again highlighting the unequal potential of the differences. However, such a role is difficult to uphold in four-cell blastomeres. species other than mice. Indeed, EGA occurs at various Lastly, without prejudice to the fundamental stages depending on the species: two-cell stage in the distinction between genuine development in utero and mouse (Flach et al. 1982) but two- to four-cell stage in models of embryonic development in vitro (blastocyst stage and derivative stem cells), we consider that it may be possible to gather some information about totipotency from those stem cells. We refer specifically to the blastomere’s ability to yield embryonic stem (ES) cells in vitro. Generally considered as pluripotent when derived the conventional way from whole blastocyst- stage embryos, ES cells seem to have an expanded potential when derived from individual blastomeres prior to the stage of ICM-TE segregation (Yang et al. 2017). Here, expanded potential means that ES cells contribute not only to the ICM after transplantation in a blastocyst, but also to the TE of chimeric embryos. We note that the derivation rate of ES cells from single mouse blastomeres correlates with the progressive loss of full developmental ability or totipotency in blastomeres isolated from various stages: 69% ES cell derivation at the two-cell stage, 40% at the early four-cell stage, 22% at the late four-cell stage and 14% at the eight-cell stage Figure 4 Possible explanations for the origin of interblastomere (Wakayama et al. 2007; Fig. 3B). differences leading to unequal potency. (A) Asynchrony of Those studies together established that blastomere developmental processes, such as mRNA degradation/deadenylation totipotency still exists after the two-cell stage, but it or EGA (embryonic genome activation) due to lack of coupling is also clear that the totipotency of single mammalian between sister blastomeres (Boni et al. 1999, Brison et al. 2014). (B) blastomeres is already no longer uniformly present at the Unequal partition at 1st mitosis: (i) incomplete diffusion of the sperm two-cell stage and it is further reduced after the two-cell content that is inherited preferentially by one blastomere (Piotrowska & Zernicka-Goetz 2002), (ii) unequal distribution of molecules such stage. This raises the question as to which mechanisms as the less abundant mRNAs (Shi et al. 2015), (iii) unequal underlie the loss of blastomere equality, leading to distribution of cytoplasmic organelles (Zheng et al. 2016, Gao et al. totipotency discontinuity. 2017), (iv) unequal segregation of chromosomes.

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Downloaded from Bioscientifica.com at 09/27/2021 07:01:46PM via free access Blastomeres: totipotent but different? R57 rat (Zernicka-Goetz 1994), four-cell stage in pigs (Hyttel a flux can become heterogeneous, causing asymmetric et al. 2000), four- to eight-cell stage in human (Braude partition of cytoplasmic components (Ajduk et al. 2011). et al. 1988), six- to eight-cell stage in rhesus monkeys In addition to this mechanism, the allocation of most (Schramm & Bavister 1999) and 8- to-16 cell stage in subcellular elements is yet to be examined in detail. cow, sheep and rabbit (Crosby et al. 1988, Plante et al. Zheng and colleagues did not find any difference in 1994, Brunet-Simon et al. 2001). In mice and humans, the number or activity of mitochondria between the a transient nuclear localization of the otherwise two 2-cell blastomeres in mice (Fig. 4Biii). We can also mitochondrial enzyme pyruvate dehydrogenase include here other elements such as the chromosomes, precedes EGA and is essential for epigenetic remodeling whose partition is controlled mainly by the spindle (Nagaraj et al. 2017), suggesting a possible metabolic assembly checkpoint (i.e. SAC). Deficiency in such regulation of EGA and perhaps also a metabolic influence a checkpoint leads to chromosome missegregation on totipotency. Hence, totipotency and its continuity causing aneuploidy (Fig. 4Biv). If such errors happen could be determined by the variability occurring in at the first division of the zygote, this immediately the regulation of major early developmental processes generates two imbalanced sister blastomeres (e.g. one as maternal mRNA degradation or/and EGA. There is a monosomic and one trisomic) that present differences of prominent regulative dimension in those scenarios. gene expression (due to gene copy number differences) A second possible cause of interblastomere differences and are limited upon separation regarding full is the unequal allocation of molecules that could be of development (monosomies are always lethal except for any kind (Fig. 4B). This unequal partition can be initiated the X chromosome, and only trisomies of chromosomes as early as the time of fertilization of the oocyte. Full 12 to 14, 16, 18 and 19 allow survival to term in mice; diffusion of the sperm content within the oocyte Gropp 1982). Unlike chromosome segregation errors at cytoplasm could be incomplete so that it is inherited the first cleavage, errors would be less detrimental when preferentially by one blastomere (Piotrowska & Zernicka- occurring at later stages, given the lower proportion Goetz 2002) (Fig. 4Bi). Even if this is still debated, sperm of cells affected. The key question is how many of the entry by itself could also contribute to the inequality cases of sister blastomere inequality can be explained between blastomeres by influencing the position of the reasonably by aneuploidy. The answer corresponds to first zygotic cleavage plan (Gray et al. 2004, Motosugi the incidence of aneuploidy in first cleavage mouse et al. 2005). Nevertheless, interblastomere differences embryos. Five different strains of mice were studied are also observed in parthenogenetic two-cell embryos, to determine that aneuploidy was observed in only which lack sperm contribution, so other mechanisms 1.1–4.3% of the embryos produced by in vivo and than those mediated by sperm entry need to be evoked in vitro fertilization after gonadotropin stimulation (Piotrowska & Zernicka-Goetz 2002). (Fraser & Maudlin 1979). Experimental abolition of As has already been mentioned, the first cleavage the SAC by reversine, a pharmacologic inhibitor of plane is critical in mediating an unequal partition of a key kinase MPS1, increased aneuploidy frequency egg content between the two-cell blastomeres. The with aneuploid blastomeres suffering impaired mitotic way this first cleavage is operated relies directly on proliferation and less developmental potential reflected the positioning of the first spindle, which needs to be in the production of fewer NANOG-positive cells (Bolton centered and oriented. The associated mechanisms et al. 2016). NANOG-positive cells are critical to build remain incompletely understood, but they seem to be the ICM at the blastocyst stage and, subsequently, the based on stochastically established forces acting on the new organism itself. In the absence of any manipulation, cytoskeleton (Chaigne et al. 2017, Salle et al. 2018). two-cell blastomeres show an asynchronous cleavage, Conversely, this leads to the hypothesis that totipotency with the faster blastomere dividing up to 1 h before the depends on the inclusion of cellular material from other one (VerMilyea et al. 2011, Balbach et al. 2012). all axial levels of the oocyte (Gardner 1996) or This gap could be related to prolonged mitosis (a sign fertilized egg (Figs 2 and 4Bii). Accordingly, this view of activated SAC), but this remains to be demonstrated considers mosaicism as the predominant concept experimentally. The percentage of aneuploid cells in versus regulation. human embryos is much higher than in mice, at least Regardless of the mosaic or regulative cause of after in vitro fertilization (Vanneste et al. 2009), and a unequal totipotency, an inevitable question is about similar situation is present in bovine (Destouni et al. the nature of the elements that need to be inherited by 2016, Tsuiko et al. 2017). the blastomeres to maintain their totipotency. They can In this paragraph, we have touched upon global be a combination of any cellular material, including mechanisms inducing inequality between blastomeres. ions, lipids, coding and noncoding transcripts, proteins, Again, we insist on the fact that the mechanisms are not cytoplasmic domains with bound transcripts and/or mutually exclusive and that they can have a different proteins as well as membranous or non-membranous impact at the molecular level. Several of the mechanisms organelles (Fig. 4B). The distribution of organelles is cited, for example, dealt with the transcripts, which mediated by actin-based cytoplasmic streaming. Such can be either degraded if they are of maternal origin or https://rep.bioscientifica.com Reproduction (2019) 158 R49–R65

Downloaded from Bioscientifica.com at 09/27/2021 07:01:46PM via free access R58 M Boiani and others produced from the new combination of chromosomes (Torres-Padilla et al. 2007, Guo et al. 2010, Burton inherited by the zygote and transmitted properly or not to et al. 2013). Arginine 26 is also a methylation substrate blastomeres, which are initiating EGA. The less abundant of PRMT4/CARM1, whose immunodetection revealed transcripts are at a higher risk of unequal partition (Shi differences between four-cell stage blastomeres (Torres- et al. 2015; further reviewed in White et al. 2018). Padilla et al. 2007). Levels of H3R2me varied between blastomeres in a similar way to H3R26me, not only in In search of molecular correlates and origins of zygotic but also in cloned mouse embryos (Liu et al. 2012). Interblastomere H3R16me2 and H3R2me differences unequal blastomere potency were specific, since the methylation of H4R3, which is Since experiments clearly demonstrated that the a target of a different methyltransferase (PRMT1), was two-cell stage blastomeres frequently exhibit different equivalent between sister blastomeres. Such specific developmental success reflecting the presence or histone modifications can impact the expression of absence of totipotency (Fig. 3A, % singlet), it should be genes encoding critical transcription factors, such as feasible to find a profile of gene expression correlating NANOG and SOX2 (Torres-Padilla et al. 2007), and, with this difference. However, instead of the two-cell potentially, the function of these factors. Consequently, stage, molecular investigations dealt firstly with the SOX2 targets, such as Sox21, are heterogeneously four-cell stage. Perhaps this preference was induced by expressed and this induces a diversification in four- the meticulous observations of Gardner, who described cell blastomere fate (Goolam et al. 2016). When the distinct categories of four-cell stage mouse embryo DNA occupancy time of SOX2 was studied along with configuration (tetrahedral, planar) with specific and OCT4 and SOX21, it was found that the DNA-binding predictable relationships to the partition of cytoplasm activity of SOX2 correlated with the allocation of the from the different positions along the animal-vegetal corresponding four-cell stage blastomeres to ICM lineage axis of the zygote (Gardner 2002). Shortly thereafter, (White et al. 2016). Those results together suggest that another study found that the progenies of two-cell ‘heterogeneous gene expression, as early as the four- stage blastomeres contribute to both embryonic and cell stage, initiates cell fate decisions by modulating extraembryonic tissues in the fetus, whereas the the balance of pluripotency and differentiation’ progenies of four-cell stage blastomeres sometimes (Goolam et al. 2016) and that ‘mammalian development contribute only to the one or the other (Fujimori et al. is to some extent mosaic from the four-cell stage onward’ 2003). This finding was also confirmed more recently (Condic 2016). (Li et al. 2017). Therefore, we will first discuss the In addition to mice, four-cell stage molecular information provided in the four-cell stage studies before diversification was also studied in other mammals. reanalyzing the data obtained at the two-cell stage. When chromatin modifications were examined in Single-cell RNA sequencing methods were exploited four-cell bovine (H3R2me2) and rabbit (acetylated in mice to allow genome-wide appreciation of histone H4K5) embryos, significant interblastomere interblastomere differences in mRNA abundance at differences were noticed, comparable with those seen the four-cell stage. After removing between-embryo in the mouse (Chen et al. 2012, Sepulveda-Rincon variation, 13 genes presented within-embryo bimodality et al. 2016). In human embryos, Chavez and colleagues (Biase et al. 2014). Transcript abundance for most of noticed marked differences in chromatin modifications these genes ranged from near zero FPKM (fragment per (H3-S28P and H4-R3me2) as early as the four-cell kilobase of transcript per million reads) in one blastomere stage (Chavez et al. 2014). At the transcript level, to 103–106 FPKM in another blastomere of the same PCR amplification ofOct4 and beta-HCG mRNA in embryo, indicating a widespread variation among sister human embryos revealed interblastomere differences, blastomeres. However, it is not yet entirely clear when suggesting that blastomere potency was not equal those differences were acquired: Investigators were able as early as the four-cell stage (Hansis 2006). Chavez to distinguish the two sister pairs forming the four-cell and colleagues confirmed that variable levels ofOct4 embryo but not which one was generated by the first- transcripts exist between blastomeres in eight-cell stage dividing vs second-dividing two-cell stage blastomere human embryos (Chavez et al. 2014). They showed (Goolam et al. 2016, Biase et al. 2018). in their study that another important marker gene of Other studies focused on histone modifications, epiblast lineage, Nanog, had abundant transcripts which can be taken as a proxy for the regulation in each blastomere of an eight-cell embryo, but was of gene expression. In four-cell stage embryos present in other cases only in two cells or even absent. exhibiting a tetrahedral shape, the signal detected by Differences in the counts of NANOG-positive cells microfluidics-based Q-PCR of the mRNA of the histone after immunofluorescence became apparent also in H3 arginine 26 methyltransferase PRDM14 or by blastocysts derived from isolated human blastomeres. immunofluorescence of H3 methylation at Arginine 26 The four blastocysts derived from a single four-cell (H3R26me2) was significantly higher in one blastomere, human embryo, for example, contained from 2 to 6 and this blastomere had its fate oriented toward ICM NANOG-positive cells (Van de Velde et al. 2008).

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In view of the above, we can draw an interim genome-wide transcriptome (respecting the original pair conclusion that blastomere diversification or departure associations of the two-cell blastomeres) would faciltate from totipotency in molecular terms is definitely the discovery of useful molecular candidates. We found supported at the four-cell stage in multiple mammalian seven studies carried out on mouse embryos but only species. Individual blastomeres from the four-cell five presented a sufficient level of linear correlation of stage embryo would then be considered insufficiently the transcriptomes within pairs to be further analyzed. regulative to fulfill the requirements of reproductive Half (50%) of the pairs considered exhibited at least a totipotency. When considering those studies, however, two-fold difference in the mRNA abundance of 17% we must also invite a related question: If the transcript/ of the genes. The expression level of only 100 (= 1%) protein level or chromatin modifications are different of the mRNAs shared by the studies was consistently among four-cell stage blastomeres, would it be possible different in four studies out of five, and only five that such interblastomere variability was already latent transcripts were found consistently differently expressed at the two-cell stage and simply became detectable at in the paired transcriptomes in all five studies, based on the four-cell stage? the same criterion as above (Tead1, 1700010D01Rik, As it has been introduced earlier, the two-cell Fstl5, Pnrc2 and Zfp472). Thus, our literature search blastomeres of the same embryo can exhibit different revealed that about 70% of sister blastomeres exhibit developmental success; therefore, there is a quest for different developmental potential, while they have only molecular elements to explain this functional difference very few transcriptomic differences (less than 1% of and, subsequently, to define a molecular signature of their commonly expressed genes) in common. In other totipotency. Embryonic stages for which some extent words, interblastomere differences identified at the of reproductive totipotency has been described (two- transcriptomic level are not reproducible from one study to-eight-cell stages) are also associated with the lowest to the other. Lack of reproducibility is a well-known complexity of blastomere transcriptome (Fig. 5). This problem (Munafo et al. 2017) and is often considered as lowest complexity was described by Piras and colleagues a criticism (claiming that it is due to, for example, poor who calculated the Shannon entropy of the transcriptome experimental design or data manipulation). However, in for mouse and human eggs and preimplantation embryos the context of this review, this low level of reproducibility (Piras et al. 2014). Therefore, it is not far fetched to may be genuine and due to multiple factors. consider that a comparison of studies that analyzed the How can low reproducibility relate to genuine data? This can happen, for example, when the origin of the trait examined is multifactorial but only some of those factors are included in the analysis. Firstly, transcripts are only one type of the possible mediators of totipotency, and protein level and localization, organelle partition, metabolic activity, and so on should also be considered. Thus, we should not expect transcripts to embody all of the interblastomere variability. Secondly, housekeeping genes, which totipotency might also rely on, were excluded from our analysis (Casser et al. 2018). Thirdly, irreproducibility might be genuine: The best case in point is the very low probability of finding an egg genetically identical to another egg, given the complexity of meiotic recombination. Lastly, the studies we analyzed worked with different mouse strains and this is known to generate additional molecular variation. Despite their small number, we ask whether we can learn something from the five gene candidates identified in all five studies or from the larger group (100 mRNAs) selected in four out of five of them. So far none of the five transcripts have been recognized as developmentally relevant genes. Nevertheless, the larger group included Eomes, Tead1, Phlda2 and Cops3, which play significant roles in cell lineage specification Figure 5 Reproductive totipotency along developmental stages is or proliferation. In fact, fetal formation in mice required inversely proportional to the complexity and variability of the a minimum of four NANOG-positive cells at the time transcriptome (Entropy H, as described in mice, see Piras et al. 2014). Data for the level of complexity and variability of the transcriptome of implantation (Morris et al. 2012), and the number of were extracted from the Piras et al. paper. Data for reproductive NANOG-positive cells might also play a comparable totipotency are taken from Fig. 3 (this paper). role in human development (Van de Velde et al. 2008, https://rep.bioscientifica.com Reproduction (2019) 158 R49–R65

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Noli et al. 2015). Thus, it could be speculated that like stem cells might be transcriptionally more similar differences in gene expression between blastomeres to blastocyst cells than blastomeres from the two-cell are conducive to a variable number of cells finally stage (Kolodziejczyk 2016). A similar approach in committed to such a NANOG-defined cell lineage (e.g. humans revealed a set of 61 genes that might constitute ICM). This is consistent with the functional observations a molecular totipotency signature distinguishing the made in MZ twin blastocysts (Casser et al. 2017). blastomeres of day 3 embryos from pluripotent cells Altogether, this would suggest that part of what makes (Galan et al. 2013). a blastomere totipotent, that is totipotency signature, Independently of the genome-wide studies reanalyzed would lie in the ability to regulate the NANOG-positive above, Zheng and collaborators found that mtrRNAs cell number (Fig. 6). located outside of mitochondria were differently Regarding the molecular definition of totipotency, abundant in the cytoplasm of sister mouse blastomeres it was also proposed that because two-cell stage at the end of the two-cell stage (Zheng et al. 2016). This blastomeres can be totipotent, the common transcripts interblastomere imbalance was observed in 10–20% between those embryos and a small subpopulation of ES of the two-cell embryos examined. When Zheng and cells with a profile of gene expression that is reminiscent colleagues tested the functional importance of 16S of the two-cell stage would correspond to the totipotency mtrRNA by gain- and loss-of-function, they found molecular signature. First, it is critical to remember that that those mtrRNAs seemed to drive the blastomere totipotent and pluripotent cells are often confused with fate toward the ICM (Zheng et al. 2016). Mechanistic stem cells, but stem cells have the property to self-renew understanding of the role of 16S mtrRNA warrants and to divide indefinitely, which is not the case for the further investigation even if it seems unlikely that a cells of the native embryo. The list of genes whose single RNA could be in charge of regulating totipotency transcripts were used to define the two-cell-like state of versus lineage commitment. ES cells and that were considered among the candidate Being aware that linear correlation is generally poor genes for totipotency markers in mice includes: MERVL, between transcript and protein level (Schwanhausser and Eif1a-like genes, Zscan4b-Zscan4f, Zfp352, et al. 2011) and that there is a two-fold variation in Tdpoz1-5 (Morgani & Brickman 2014, Wu et al. 2017). the amount of protein and nucleic acid contained in Those genes were not part of the common differences the different blastomeres of an individual eight-cell found between blastomere at the two-cell stage (Casser mouse embryo (Cheung et al. 2013), the single-cell et al. 2018), simply because selection criteria (two-fold proteome analysis of sister blastomeres is likely to be difference of mRNA level in at least 50% of the pairs) a key experimental approach to capture the molecular were not fulfilled. Functionally, two cell-like stem cells determinants of blastomere totipotency (Virant-Klun can contribute to both embryonic and extraembryonic et al. 2016). Older and more recent studies contribute lineages in chimeras; therefore, they could be called data supporting interblastomere differences at the protein ‘totipotent’ according to the more permissive definition level, for example, SNAI1/SNAI2 (Bell & Watson 2009) of totipotency, which is expressly not taken as the default and GADD45a (Rosas et al. 2018). It will be necessary to in this review. Moreover, in global terms, the two-cell- analyze the composition of each blastomere at the two- cell stage and identify critical proteome requirements supporting totipotency, in order to make further progress, but, of course, it is impossible to combine a destructive assay with a proof of function for the same cells. Cellular biopsy may help, but because we suspect a dose of mosaicism, removing a subcellular volume of the blastomeres for analysis does not appear a safe way to infer the average blastomere composition. An interim solution to find the molecular origin of the early diversification occurring between two-cell blastomeres in 70% of the embryos could be to use modeling of the proteomic data collected from reproducibly bisected oocytes (i.e. along the animal-vegetal axis) or from single Figure 6 Developmental view of totipotency-pluripotency transition. blastomeres whose relationship to the orientation of the Totipotency is an important characteristic of the fertilized egg or zygotic cleavage plane is known. A comparison between zygote that is conserved or lost partially/totally with the first cleavage the daughter cells obtained from the two-cell blastomere and subsequent embryonic divisions. Embryonic cells evolve to that cleaved first and second could also be productive. pluripotency, as observed in stem cells, exhibiting several properties Transcriptomic datasets for all blastomeres of the same such as self-renewal and high differentiation capacity. Reproductive totipotency is particularly associated with the later production of a four-cell embryo are available (Goolam et al. 2016, sufficient number of NANOG-positive cells in the stem cell Biase et al. 2018), but their origin from first-divided or compartment of the epiblast (Morris et al. 2012). second-divided two-cell blastomere was not recorded.

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Moreover, the fact that cleavage axes have different maintain totipotency in both blastomeres, because (1) orientations in different embryos could result in multiple the sheltered environment of the mother makes cell ways of maternal molecule partition, compromising the damage improbable and thereby a second totipotent reproducibility and comparability of these data. blastomere – to compensate for damage – superfluous and (2) the natural tendency of increase in entropy is Conclusions and revised model: mosaic better satisfied when the sister blastomeres are not equal. In an evolutionary perspective, this inequality is also a construction of zygotic totipotency source of diversity, which is a substrate of Darwinian In this review, we revisited the multifaceted concept selection. Accordingly, it is tempting to consider that of totipotency, starting from the historical notions the maintenance of totipotency through cleavage is no and confronting them with the data available on the developmentally programmed event, and that there development of MZ twins and gene expression profiles is no preordained mechanisms to ensure totipotency of sister blastomeres, to achieve a modern integrative in both blastomeres. As a result, either both or just view. Among various definitions, we specifically defined one could be totipotent, fortuitously, depending for totipotency as the ability of one cell to give rise to a new instance on how the randomly oriented axis of the first complete organism. It should be underlined that this zygotic cleavage (Louvet-Vallee et al. 2005, Zernicka- definition overlaps with the definition of developmental Goetz 2005) apportions some spatially restricted biology itself (study of processes by which a one-cell developmental information laid in the ooplasm. This stage embryo or zygote develops into a fully structured does not exclude cases where both blastomeres are organism). Thus, totipotency is a key concept in our not totipotent and the embryo is, therefore, unviable. understanding of the developmental program through This can be due to the failure of both blastomeres which metazoan organisms are generated. reflecting errors of chromosome segregation at In mammals, it was generally accepted that early first zygotic mitosis (most aneuploidies preclude blastomeres were equally (toti)potent before showing development to term; Rabinowitz et al. 2012, Babariya overt signs of molecular differentiation at the four-cell et al. 2017) or the lack of developmental competence stage (Torres-Padilla et al. 2007). However, our review of some oocytes, i.e. not all of them developed after documented that only about one-third of two-cell embryos fertilization because of a deficiency in maternal factors achieve the full development of monozygotic twins after (Christians 2017). Furthermore, cases where both experimental separation of the blastomeres performed blastomeres are not totipotent can also be consistent in various mammalian species (mouse, cow, sheep, rat with unequal segregation of determinants or ingredients and monkey). It is important to stress ‘full development’ that should be inherited in a defined amount by the (to term), which is the compass we used throughout the sister two-cell blastomeres to support their totipotency. review. Hence, two-cell blastomeres have a common Although we need to exercise caution about these disposition to form an organism, but its extent is not determinants, because segregation of proven ones has necessarily equal. This pattern would go unnoticed in yet to be documented in mammalian embryos and the intact embryo, because the ‘superior’ or more potent histochemical diagnostic methods have limitations blastomere would mask the deficit of the other blastomere (Littwin & Denker 2011), we are not totally clueless. (provided the deficit is relevant for development). As a first As mentioned earlier, totipotency of a blastomere in conclusion of this review, we should recognize that most mice, for example, could correlate with a number of ≥4 mammalian two-cell embryos have an inner imbalance epiblast cells formed by the blastomere progeny at the of totipotency between the sister blastomeres and that this time of implantation (Morris et al. 2012) and, therefore, does not impair their development as a whole. Given the rely on some determinants regulating the accumulation numbers we captured in this review (thousands of two- of a blastomere’s progeny cells in the epiblast (e.g. cell embryo bisections and hundreds of embryo transfers), NANOG). This leads to the second conclusion of the first conclusion seems rather solid, and we consider this review: The molecular signature of totipotency that further research would be more productive if used is obviously complex, yet the search for it might be not to double-check whether the imbalance really exists, facilitated if it is oriented toward the determination of but to illuminate its molecular nature. the epiblast lineage (Fig. 6). To acknowledge this imbalance is to recognize Based on the findings reviewed above, we suggest a that there is no strict maintenance of totipotency at or revised model of mammalian totipotency discontinuity, beyond the two-cell stage, and this raises the question where mosaicism and imperfect regulation (imperfect whether the loss is regulated or stochastic. We posit because it is unable to offset the initial difference) are that a regulated process would have been reflected in sequentially involved. Only the zygote is totipotent with a more homogeneous pattern of molecular differences certainty in the mammalian life cycle. Its organization is between blastomeres than the pattern we actually mosaic, so that the first cleavage axis divides still elusive saw. In teleological terms, we consider that there is no determinants between the two-cell stage blastomeres, compelling reason for mammalian two-cell embryos to which can – but do not have to be – both totipotent. https://rep.bioscientifica.com Reproduction (2019) 158 R49–R65

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