© 2020. Published by The Company of Biologists Ltd | Development (2020) 147, dev190629. doi:10.1242/dev.190629

REVIEW Mechanisms of human development: from cell fate to tissue shape and back Marta N. Shahbazi*

ABSTRACT activated ion channels (Coste et al., 2010), mechanosensitive Gene regulatory networks and tissue morphogenetic events drive the transcription factors (Dupont et al., 2011) or directly by the nucleus emergence of shape and function: the pillars of embryo development. (Kirby and Lammerding, 2018). Once sensed, mechanical cues – Although model systems offer a window into the molecular biology of are transduced into biochemical signals a process known as cell fate and tissue shape, mechanistic studies of our own mechanotransduction (Chan et al., 2017). The conversion of development have so far been technically and ethically challenging. mechanical cues into biochemical signals leads to changes in However, recent technical developments provide the tools to gene expression and protein activity that control cell behaviour, cell describe, manipulate and mimic human in a dish, thus fate specification and tissue patterning. opening a new avenue to exploring human development. Here, I Current consensus focuses on two main ideas to explain the discuss the evidence that supports a role for the crosstalk between emergence of tissue patterns in response to morphogen (see Glossary, ‘ ’ cell fate and tissue shape during early human embryogenesis. This is Box1)signals.Inthe positional information model (Wolpert, a critical developmental period, when the body plan is laid out and 1969), the concentration of a morphogen serves as a coordinate of the many pregnancies fail. Dissecting the basic mechanisms that position of a cell within a tissue. Cells respond to a specific coordinate cell fate and tissue shape will generate an integrated morphogen concentration making a specific fate choice. Therefore, understanding of early embryogenesis and new strategies for an existing asymmetry is transformed into a pattern of cell fates. On therapeutic intervention in early pregnancy loss. the contrary, in the reaction-diffusion model (Turing, 1952), pattern formation is a self-organising phenomenon. The basic idea behind KEY WORDS: Human embryo, Morphogenesis, Cell fate, Embryonic this model is the presence of a short-range activator that induces its stem cells, Aneuploidy, Polarisation own expression and the expression of a long-range inhibitor. A spontaneous increase in the local concentration of the activator (a Introduction symmetry-breaking event) will induce a further increase in the levels Developmental biologists are faced with a question of form and of the activator and expression of the inhibitor. However, as the function. Starting from a relatively simple spore, seed or egg, inhibitor has a higher diffusion rate, this will lead to a peak of multiple cell types arise that perform specific functions. These activation surrounded by a valley of repression. These two different cells organise into diverse configurations, leading to the mechanisms, positional information and reaction-diffusion, co-exist emergence of tissues of a defined form. Development requires during development to generate patterns (Green and Sharpe, 2015). mechanical changes in cell and tissue shape, which drive The use of model organisms allows us to explore the mechanisms morphogenesis, and gene expression changes, which regulate cell that orchestrate the crosstalk between cell fate and tissue shape. For fate decisions and tissue patterning. These two developmental example, cytoskeletal forces lead to the acquisition of cell polarity processes are not independent, as changes in cell fate can modify the and the differential segregation of cell fate determinants in architecture of tissues, and vice versa (Chan et al., 2017). C. elegans (Goehring et al., 2011), an emerging lumen traps Mechanochemical feedback at the molecular, cellular and tissue (FGF) molecules, which lead to levels coordinates this crosstalk and instructs tissue self-organisation differentiation in the lateral line primordium of zebrafish (Durdu (see Glossary, Box 1) (Hannezo and Heisenberg, 2019). et al., 2014), and tissue deformations control midgut differentiation Molecular motors generate mechanical forces that are transmitted in Drosophila embryos (Desprat et al., 2008). As human beings, our across tissues via the cytoskeleton and cell-cell adhesion proteins own development remains the most significant to study, yet is still (Heisenberg and Bellaiche, 2013). In the embryo, this leads to mysterious due to technical and ethical limitations (Box 2). In this changes in cell shape and position, to large tissue-scale Review, I draw upon knowledge gained from studies in model deformations, and consequently to morphogenesis. Cells sense organisms, embryonic research and human to mechanical cues – a process termed mechanosensation – via propose mechanistic models of three critical developmental events: extracellular matrix (ECM) adhesion proteins (Schwartz, 2010), compaction and polarisation at the stage; embryonic cell-cell adhesion proteins (Benham-Pyle et al., 2015), stretch- epithelialisation at the time of implantation; and pluripotent cell differentiation at (Fig. 1). The emerging picture supports a role for the crosstalk between tissue shape and cell fate MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge as a determinant of human embryogenesis. Biomedical Campus, Cambridge CB2 0QH, UK.

*Author for correspondence ([email protected]) Compaction and polarisation: the first morphogenetic transformation M.N.S., 0000-0002-1599-5747 Compaction This is an Open Access article distributed under the terms of the Creative Commons Attribution At the 8- to 16-cell stage, human embryos undergo the process of License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. compaction; (see Glossary, Box 1) adhere tightly to DEVELOPMENT

1 REVIEW Development (2020) 147, dev190629. doi:10.1242/dev.190629

Box 1. Glossary Box 2. Historical perspective of human embryo . An extra-embryonic membrane that protects the development developing foetus from mechanical damage and the maternal The birth of human embryology as a scientific discipline is intimately immune response. linked to the creation of human embryo collections (Yamada et al., 2015; . A clade of vertebrates in which the embryo develops while Gasser et al., 2014). The pioneering work of Franklin Mall led to the being protected by extra-embryonic membranes, including the amnion. It creation of the Carnegie collection in 1887, which harbours more than encompasses , and . 10,000 human embryo specimens, and established the basic staging Amniotic cavity. Fluid-filled sac located between the amnion and the criteria for the developmental classification of human embryos (Keibel embryo, the function of which is to protect the embryo from mechanical and Mall, 1912). Other collections were later created, such as the Kyoto damage and the maternal immune system. collection, which today holds ∼44,000 specimens (Nishimura et al., . Fluid-filled cavity present in stage embryos. 1968). Much of our current textbook knowledge of human development is Blastocyst. An embryo composed of an outer trophectoderm layer, an derived from the early descriptive studies of these samples. and an internal blastocoel cavity. The development of in vitro (IVF) of human eggs initiated a Blastomeres. Cells of the early embryo, formed by cleavage of the revolution in human embryo and stem cell research and human . reproduction (Edwards et al., 1969; Rock and Menkin, 1944; Shettles, . Trophectoderm stem cell compartment of the human 1955). This initial milestone was followed by the development of embryo. It plays a key role during blastocyst implantation and it generates conditions to culture fertilised human eggs in vitro for up to 5-6 days differentiated trophectoderm cells such as and (Edwards et al., 1970; Steptoe et al., 1971), and ultimately led to the birth extravillous . of the first IVF baby in 1978, thanks to the tireless efforts of Robert . Outermost primary that gives rise to the nervous Edwards, Patrick Steptoe and Jean Purdy. Since then, the field of human system, the epidermis and its appendages. embryology has flourished. IVF has allowed scientists to describe the Embryonic genome activation. The process whereby zygotic dynamics of key morphogenetic processes during early human transcription is initiated and takes over the maternally stored mRNAs development, such as cleavage, compaction and (Wong and proteins. In human embryos, it takes place at the four- to eight-cell et al., 2010; Marcos et al., 2015; Iwata et al., 2014); to characterise cell stage transition. lineage specification events by studying the transcriptional and Embryoid body. Three dimensional aggregates of pluripotent stem cells epigenetic profiles of all the cells present in a developing human commonly used to study differentiation. embryo (Niakan and Eggan, 2013; Petropoulos et al., 2016; Braude . Innermost primary germ layer that gives rise to the et al., 1988; Zhu et al., 2018); to identify genetic and chromosomal epithelial cells of the gastrointestinal, respiratory, endocrine and abnormalities that compromise human embryo development (Munne urinary systems. et al., 2009; Vanneste et al., 2009); and, perhaps more importantly, to . The pluripotent embryonic tissue that gives rise to germline establish human lines (Thomson et al., 1998), which and soma, as well as the extra-embryonic amnion. on their own have revolutionised our approach to studying human Epithelial-to-mesenchymal transition. Cellular program that leads to development and devising regenerative therapies. However, until the loss of epithelial features (i.e. polarised organisation and cell-cell recently, gene function could not be studied in the context of human adhesion) and the acquisition of a mesenchymal phenotype. It plays a embryos. The recent generation of OCT4 knockout human embryos key role during gastrulation. represents a turning point in the field (Fogarty et al., 2017). This study Extra-embryonic ectoderm. Trophectoderm-derived tissue that forms highlighted differences in gene function between mouse and humans, during early post-implantation in mouse embryos. and established a gold standard for functional studies in human Inner cell mass. A group of cells located inside the blastocyst that gives embryos. Thus, human embryology is becoming an experimental rise to the epiblast and the . science; I argue that, in the years to come, we will witness a surge in Morphogen. A signalling molecule that determines cell fates in a the number of mechanistic studies exploring our own development. concentration-dependent manner. . Primary germ layer positioned in between the ectoderm and the endoderm. It gives rise to muscle, bone, connective tissue, blood vessels, red and white blood cells, and the mesenchyme of various each other, maximising their cell-cell contact area, which leads the organs such as skin, or gonads. embryo to acquire a compressed morphology. This process is Primary . Extra-embryonic membrane formed by hypoblast conserved in all mammalian species studied so far, but the timing cells around 7 to 8 days post-fertilisation. By E12-15, it gives rise to a varies. The precise timing of the onset and completion of secondary yolk sac, the primary functions of which are hematopoiesis compaction is important for the successful development of both and nutrient transport. Primitive endoderm (mouse) or hypoblast (human). An extra- mouse and human embryos (Mizobe et al., 2017; Kim et al., 2017; embryonic tissue characteristic of pre-implantation that Skiadas et al., 2006). Human embryos in which compaction is either gives rise to the yolk sac. initiated prematurely or delayed have a lower potential to form . Transient structure that appears in the posterior blastocysts (see Glossary, Box 1). Premature compaction is epiblast and marks the start of gastrulation. associated with cytokinetic failure and multi-nuclear blastomeres Primordial germ cells. Specialised cells, precursors of the gametes, (Iwata et al., 2014), whereas delayed compaction is associated with that are specified in the epiblast of mammalian embryos during early post-implantation development. a reduced number of inner cell mass (ICM) cells (see Glossary, Pro-amniotic cavity. Luminal cavity that is formed in the mouse epiblast Box 1) (Ivec et al., 2011). at E5.0. The fusion between the epiblast and extra-embryonic ectoderm The mechanisms that drive compaction in human embryos cavities leads to the formation of the amniotic cavity by E5.75 in mouse remain unknown and, instead, have mainly been explored using embryos. mouse embryos. The key players are the actomyosin cytoskeleton Self-organisation. Spontaneous generation of ordered structures that and the cell adhesion protein E-cadherin. The actomyosin results from the interaction between elements that have no previous pattern. cytoskeleton undergoes pulsed contractions that generate force Sialomucins. Glycosylated proteins of the mucin family that contain the and increase membrane tension. This happens specifically at the acidic sugar sialic acid. cell-free surface of the embryo, as E-cadherin keeps contractility Trophectoderm. An extra-embryonic tissue present in pre-implantation low at cell-cell adhesion sites (Maitre et al., 2015). An alternative embryos that gives rise to the . model proposes that E-cadherin-containing filopodia are the major

drivers of compaction. By binding to and promoting elongation of DEVELOPMENT

2 REVIEW Development (2020) 147, dev190629. doi:10.1242/dev.190629

Human developmental time 0 1 2 3 4 6 10 14 80

Pre-implantation Naive Pluripotency loss Embryonic Amnion lineage pluripotency and lineage genome activation specification Fate specification exit commitment Epithelial-to- Compaction Inside cell Epiblast Amniotic cavity mesenchymal and polarisation generation epithelialisation formation transition Shape

Implantation Amnion

Amniotic Primary Amnion Primitive streak Blastocoel cavity yolk sac Yolk sac Implantation Pro-amniotic Placenta cavity

Mouse developmental time

0 1.5 2 2.5 3 4 5.5 6.5 9.5

Pre-implantation Naive Pluripotency loss Embryonic lineage pluripotency and lineage genome activation Fate specification exit commitment Pro-amniotic Epithelial-to- Compaction Inside cell Epiblast cavity mesenchymal and polarisation generation epithelialization formation transition Shape

Fig. 1. Overview of human and mouse embryo development. Upon fertilisation, mouse and human embryos undergo a series of cleavage divisions. The embryonic genome becomes activated by the two-cell stage in mouse embryos and at the four/eight-cell stage transition in human embryos. It is followedby compaction and polarisation, which occur at the eight-cell stage in mouse embryos, and between the eight- to 16-cell stage in human embryos. Formationofa hollow cavity, the blastocoel, denotes the formation of the blastocyst, which, by embryonic day E4 (mouse) and E6 (human), is composed of three main tissues: epiblast, hypoblast and trophectoderm. Upon implantation (E5 in mouse and E7 in human), embryos undergo a global morphological transformation. The embryonic epiblast loses its naïve pluripotent character, becomes epithelial and forms the pro-amniotic cavity (mouse), which spans both the epiblast and the trophectoderm-derived extra-embryonic ectoderm, and the amniotic cavity (human). A key difference between mouse and human embryos relates to the formation of the amnion. Whereas in mouse embryos amnion formation takes place during gastrulation, in human embryos a subset of epiblast cells differentiates to form the squamous amniotic epithelium during early post-implantation (E10). As a result, the human epiblast acquires a disc shape and the mouse epiblast a cylindrical morphology. On embryonic days 6.5 (mouse) and 14 (human), gastrulation is initiated in the posterior epiblast. Cells undergo an epithelial-to- mesenchymal transition, lose their pluripotent character and commit to one of the germ layers. Epiblast-derived tissues are shown in pink, hypoblast-derived tissues are shown in blue and trophectoderm-derived tissues are shown in green. the apical surface of neighbouring blastomeres, they trigger the cell protein kinase C (PKC) activation at the eight-cell stage triggers shape changes that take place during compaction in a non-cell- actomyosin polarisation via RhoA (Zhu et al., 2017). In addition, autonomous manner (Fierro-Gonzalez et al., 2013). Whether these transcription is required for Par complex polarisation. The mechanisms are active in human embryos remains unknown. In transcription factors Tfap2c and Tead4 are expressed after terms of timing, studies in mouse embryos suggest that the onset of embryonic genome activation (EGA, see Glossary, Box 1) and compaction could be controlled by the decrease in the nuclear/ induce the expression of several regulators of the actin cytoskeleton, cytoplasmic ratio that takes place during the early cleavage divisions which become progressively upregulated from the two- to eight-cell and/or the dilution of a potential inhibitor of compaction present in stage. This leads to a remodelling of the actin network that is the zygote (Kojima et al., 2014). These are interesting hypotheses necessary for the clustering of apical polarity proteins, and that require further experimental validation. culminates with the formation of the mature apical domain. As a result, the combined artificial activation of RhoA and expression of Polarisation Tfap2c and Tead4 is sufficient to induce premature embryo Concomitant with the process of compaction, cells acquire polarisation and to advance morphogenesis (Zhu et al., 2020). apicobasal polarity. In mouse embryos, the basolateral domain Studies in human embryos revealed the presence of apical concentrates cell adhesion proteins, such as E-cadherin, whereas the microvilli and basolateral E-cadherin by the time embryos compact apical domain is enriched in apical polarity proteins, such as the (Alikani, 2005; Nikas et al., 1996), but the exact sequence of events partitioning defective (Par) complex and cytoskeletal components and the mechanisms leading to human embryo polarisation remain

(Leung et al., 2016). Recent studies in mice have revealed that to be explored. Given that EGA precedes polarisation, it is tempting DEVELOPMENT

3 REVIEW Development (2020) 147, dev190629. doi:10.1242/dev.190629 to speculate that, as in mouse embryos, EGA controls the timing of in actomyosin contractility and tension between polar and apolar human embryo polarisation via as yet unknown transcription factors cells also regulate Yap localisation (Maitre et al., 2016). (Zhu et al., 2020) (Fig. 2). Our knowledge of lineage specification in human embryos remains rudimentary. Single-cell sequencing analyses have The first cell fate decision generated a spatiotemporal catalogue of gene expression and have Polarisation is fundamental for the divergence of embryonic and revealed a number of transcriptional differences between mouse and extra-embryonic lineages in mouse embryos. In support of this human embryos (Niakan and Eggan, 2013; Blakeley et al., 2015; notion, loss-of-function studies demonstrate that Par complex Petropoulos et al., 2016; Yan et al., 2013). At the compacted components are needed for the acquisition of an extra-embryonic stage (embryonic day E4), the transcription of TE genes such as trophectoderm (TE) (see Glossary, Box 1) fate (Plusa et al., 2005; GATA3 and PDGFA is initiated, although cells still express ICM Alarcon, 2010). Cells that inherit the apical domain differentiate genes (Petropoulos et al., 2016). It is not until the early blastocyst towards TE, whereas cells that do not inherit the apical domain and stage (embryonic day E5) when lineage-specific markers start to are internalised become the ICM, and hence the precursors of the become mutually exclusive. Aggregation experiments have shown embryonic epiblast (see Glossary, Box 1) and extra-embryonic that E5 TE cells still retain the ability to form ICM cells (De Paepe primitive endoderm (PE) (Korotkevich et al., 2017) (see Glossary, et al., 2013), and, conversely, isolated human ICMs can also Box 1). Inner cell allocation is mainly regulated by apical generate TE cells (Guo et al., 2020). These results indicate that, even constriction, heterogeneities in cortical tension and, to a lesser if the ICM-TE lineage segregation takes places at the blastocyst extent, by the orientation of cell division (Samarage et al., 2015; stage (Petropoulos et al., 2016), cells are not yet fully committed. Anani et al., 2014). This leads to the first spatial segregation of cells Analysis of protein expression and localisation has also in the mouse embryo: outer polarised cells and inner apolar cells. uncovered interesting differences between mouse and human Therefore, two morphogenetic cues, polarity and position, regulate embryos. YAP localises to the nucleus both in TE and epiblast the first cell fate decision in mouse embryos. cells in human blastocysts (Qin et al., 2016), but becomes restricted Mechanistically, the transcription factor Yap is responsible for to the TE at the late blastocyst stage (Noli et al., 2015), in agreement the ICM-TE bifurcation in mouse embryos (Sasaki, 2015). In with a delayed ICM-TE segregation in human embryos when polarised cells, Yap translocates to the nucleus and binds the compared with mouse. Supporting a potential role for YAP during transcription factor Tead4, which leads to the expression of TE TE specification, loss of YAP prevents human embryonic stem cell genes such as Cdx2 (Rayon et al., 2014). In turn, Cdx2 inhibits the (ESC) differentiation to the TE fate (Mischler et al., 2019; Guo expression of the ICM-specific pluripotency markers Oct4 (Niwa et al., 2020). Surprisingly, CDX2, a master TE regulator in mouse et al., 2005) and Nanog (Strumpf et al., 2005). By the 32-cell stage, embryos, is not expressed until the late blastocyst stage in human Cdx2+ cells are irreversibly committed to the TE fate (Posfai et al., embryos (Niakan and Eggan, 2013); its target genes in the mouse, 2017; Nichols and Gardner, 1984; Gardner, 1983). Yap localisation Eomes and Elf5, are not expressed in pre-implantation human TE is regulated by several upstream factors. In apolar cells, the Hippo (Blakeley et al., 2015); and loss of OCT4 in human embryos leads to pathway component angiomotin activates the Hippo kinase Lats, a downregulation of CDX2 expression (Fogarty et al., 2017), which phosphorylates Yap, leading to its cytoplasmic sequestration. indicating that, in contrast to mouse embryos, OCT4 and CDX2 do In polarised cells, angiomotin is recruited to the apical domain and not mutually repress each other in human embryos (Niwa et al., becomes inactivated, allowing Yap to translocate to the nucleus 2005; Ralston et al., 2010). Globally, these findings raise the (Leung and Zernicka-Goetz, 2013; Hirate et al., 2013). Differences possibility that different transcription factors control the

Cell fate: Morphogenesis: gene expression changes tissue shape changes

Embryonic genome Polarisation activation ? TranscriptionTranscription CytoskeletalCytoskeletal Apical polarity Maternal Embryonic proteinsproteins proteins RNA factors genes ffactorsactors

Time Eight-cell stage Four-cell stage ? Nuclear YAP Trophectoderm specification


Fig. 2. Proposed cell fate-tissue shape crosstalk during human pre-implantation development. The onset of apicobasal polarisation at the eight-cell stage may be controlled by the expression of embryonic genes, potentially encoding transcription factors. As recently shown in mouse embryos, embryonic genome activation induces the expression of regulators of the actin cytoskeleton. This leads to a remodelling of the actin network that is necessary for the clustering of apical polarity proteins, and culminates in the formation of the mature apical domain. In turn, the acquisition of apicobasal polarisation may lead to nuclear YAP localisation, as seen in the mouse, and expression of trophectoderm-specific transcription factors. This model remains to be functionally validated in human embryos. Question marks denote molecular connections that have not been validated in human embryos. DEVELOPMENT

4 REVIEW Development (2020) 147, dev190629. doi:10.1242/dev.190629 specification of human TE (Fig. 2). GATA3 is an interesting Agerholm et al., 2019), although this hypothesis remains to be candidate, as its downregulation in primate embryos inhibits TE formally validated. differentiation (Krendl et al., 2017) and because it is a downstream A recent study in mouse embryos revealed an unexpected role for target of TEAD4/YAP (Home et al., 2012) that inhibits OCT4 the blastocoel during epiblast-PE specification (Ryan et al., 2019). expression (Krendl et al., 2017) in human ESCs differentiated to TE. The authors propose that lumen expansion is needed for correct Future studies are needed to determine whether YAP and GATA3 lineage segregation (Ryan et al., 2019). Interestingly, in isolated play a role in TE specification in human embryos. This is especially ICMs, epiblast and PE progenitors segregate, although with an important as protocols to differentiate human ESCs into TE may not increased proportion of PE to epiblast cells (Wigger et al., 2017). reflect the in vivo specification route. Differentiation of human This result suggests that, although the second cell fate decision can ESCs upon addition of BMP4 has been widely used to obtain TE- take place in the absence of the blastocoel, it may be required to fine- like cells (Amita et al., 2013; Xu et al., 2002). However, various tune the relative numbers of epiblast and PE progenitors. reports suggest that cells acquire an extra-embryonic and embryonic mesoderm and/or extra-embryonic amniotic fate rather than a TE Human embryo culture beyond implantation fate (Guo et al., 2020; Bernardo et al., 2011). Although new Our knowledge of human embryo development at and beyond differentiation protocols are being developed (Guo et al., 2020; implantation (E7) is very scarce. It has been mainly limited to the Mischler et al., 2019; Horii et al., 2019; Dong et al., 2020), these do analysis of human embryo specimens from the Carnegie collection not reflect the 3D organisation of the embryo, and the potential (Box 2), which have revealed that implanting blastocysts undergo contribution of embryo polarisation and cell position to TE global morphological remodelling to form a disc-shaped embryo specification cannot be explored. Therefore, the molecular players (Hertig et al., 1956). Conventional human embryo culture methods involved in human TE specification, and whether, as in the mouse, only allow embryo growth and survival up to E6-7 (late blastocyst polarisation controls the first lineage segregation in human embryos stage). Co-cultures with endometrial cells have traditionally been remain to be determined. used to explore the crosstalk between the embryo and the uterus (Weimar et al., 2013; Lindenberg et al., 1985), but whether the The implanting embryo: a global transformation embryo undergoes proper morphogenesis in this setting is unclear. The second cell fate decision Recently, human embryos have been cultured up to day 13 in vitro, Human and mouse embryos reach the blastocyst stage at embryonic thus permitting insight into the major morphological days E5 and E3.5, respectively. Blastocyst formation involves a transformations of post-implantation human development – TE process of to form the blastocoel (see Glossary, Box 1). In differentiation (West et al., 2019), amniotic cavitation (see Glossary, mouse embryos, blastocoel formation is driven by the accumulation Box 1) and primary yolk sac (see Glossary, Box 1) formation – in of pressurised fluid between cells. This fluid moves from the outside the absence of interactions with maternal tissues (Deglincerti et al., medium into the intercellular space as a consequence of an osmotic 2016; Shahbazi et al., 2016). This culture method has been taken gradient, leading to the formation of microlumens that coarsen to forward by the addition of a 3D matrix, which allows amnion (see form a single lumen (Dumortier et al., 2019). In turn, luminal Glossary, Box 1) specification and further development up to the pressure leads to increased cortical tension in the TE and tight gastrula stage (Xiang et al., 2019). These systems open the door to junction maturation to allow lumen expansion (Chan et al., 2019). study human embryo development beyond implantation; e.g. they This is a perfect example of how mechanical cues, in this case have been used to generate a single-cell transcriptome and hydrostatic pressure, affect differentiation (Chan and Hiiragi, 2020). methylome map of post-implantation human embryos (Zhou Once the embryo reaches the blastocyst stage, it is ready to et al., 2019; Xiang et al., 2019). But given the lack of an in vivo implant in the maternal uterus. At this stage, mouse and human reference, comparisons with primate embryos are important for embryos undergo a second cell fate specification event: the ICM validation (Nakamura et al., 2016; Boroviak and Nichols, 2017; Ma segregates into pluripotent epiblast and extra-embryonic hypoblast et al., 2019; Niu et al., 2019). (see Glossary, Box 1). Whereas in mouse embryos the specification of TE cells precedes the segregation of ICM cells into epiblast and Epiblast epithelialisation and amniotic cavitation PE (the mouse equivalent of the human hypoblast), the timing of The first transformation that epiblast cells undergo is the formation epiblast-hypoblast segregation in human embryos is still under of an epithelial tissue that lines the amniotic cavity. This process is debate. Although some studies report a concomitant specification of evolutionarily conserved in all amniotes (see Glossary, Box 1) and the three lineages, epiblast, hypoblast and TE, in human embryos is a fundamental prerequisite for gastrulation, lineage specification (Petropoulos et al., 2016), others have identified an early ICM state and developmental progression (Sheng, 2015). Mutations that that precedes the epiblast-hypoblast split (Stirparo et al., 2018). impair pro-amniotic cavity (see Glossary, Box 1) formation lead to Upon specification, human hypoblast cells become apicobasally developmental arrest in mouse embryos and failed differentiation in polarised and express apical proteins such as the sialomucin (see embryoid bodies (see Glossary, Box 1) (Sakai et al., 2003; Liang Glossary, Box 1) protein podocalyxin (Shahbazi et al., 2017). In et al., 2005; Smyth et al., 1999). mouse embryos, polarisation is required for PE maintenance. In mouse and human embryos, epiblast epithelialisation takes Inhibition of atypical protein kinase C (aPKC), a component of the place during implantation (Shahbazi and Zernicka-Goetz, 2018). In Par complex, or β1-integrin, a cell-ECM adhesion protein, leads to response to ECM proteins secreted by the extra-embryonic tissues defects in epithelialisation and PE maturation (Saiz et al., 2013; Liu (Li et al., 2003), epiblast cells become polarised and form a rosette- et al., 2009). Studies using human ESCs suggest that aPKC may like structure that undergoes lumenogenesis to form the amniotic also control the epiblast-hypoblast lineage segregation. Inhibition of cavity (Bedzhov and Zernicka-Goetz, 2014; Luckett, 1975). In the aPKC is needed to maintain naïve pre-implantation-like pluripotent mouse, epiblast polarisation is triggered by β1-integrin signalling human ESCs (Takashima et al., 2014). These cells are competent to and requires phosphatase and tensin homologue (Pten) and integrin- differentiate into extra-embryonic endoderm cells, and this has been linked kinase (Ilk) activity (Meng et al., 2017; Sakai et al., 2003), proposed to be dependent on aPKC signalling (Linneberg- whereas lumenogenesis is triggered by exocytosis of apical vesicles DEVELOPMENT

5 REVIEW Development (2020) 147, dev190629. doi:10.1242/dev.190629 and membrane separation (Shahbazi et al., 2017), a process known as Experiments in human ESCs revealed that epithelialisation leads hollowing (Bryant and Mostov, 2008). In the human context, further to decreased pluripotent gene expression via integrin β1 activation mechanistic insight has been gained through the use of 3D ESC (Hamidi et al., 2020), and therefore epiblast epithelialisation could cultures, which recapitulate the processes of epithelialisation and facilitate pluripotency exit in vivo. In mouse ESCs, a decrease in cavity formation (Shahbazi et al., 2016; Taniguchi et al., 2015). membrane tension is necessary for naïve pluripotency exit (Bergert Whereas actin polymerisation promotes cavity formation, actomyosin et al., 2019), as it allows the activation of FGF signalling, a trigger of contractility and the activity of Rho-associated protein kinase (ROCK) naïve exit, via endocytosis of FGF receptors (De Belly et al., 2019). prevent lumenogenesis (Taniguchi et al., 2015). Accordingly, the In human ESCs FGF signalling inhibition is implied as a key addition of a ROCK inhibitor to human and monkey embryo culture requirement for naïve human ESCs, and its activation is associated promotes amniotic cavity formation (Xiang et al., 2019; Niu et al., with naïve pluripotency exit (Di Stefano et al., 2018). Thus, it is 2019). The positive effect of ROCK inhibition during lumenogenesis tempting to speculate that loss of the naïve state in human embryos has been described in other models of hollowing (Bryant et al., 2014), could also be regulated by changes in membrane tension leading to suggesting it is evolutionarily conserved. FGF receptor endocytosis (Fig. 3). Future studies are needed to Once the incipient amniotic cavity is formed, mouse and human determine whether similar changes in membrane tension and embryos acquire different morphologies (Fig. 1). In the mouse signalling are observed beyond 2D cultures, using systems that embryo, the TE-derived extra-embryonic ectoderm also undergoes a recapitulate both epiblast gene expression and shape. Cell-cell and cavitation event. The subsequent fusion of the epiblast and extra- cell-matrix interactions, as well as the mechanical properties of the embryonic ectoderm cavities leads to the formation of the pro- ECM, are major regulators of pluripotent stem cell identity in vitro amniotic cavity, which spans both tissues (Christodoulou et al., (Ranga et al., 2014; Przybyla et al., 2016; Pieters and Van Roy, 2014; 2018). In contrast, the human TE gives rise to the cytotrophoblast Hamidi et al., 2020). Upon naïve pluripotency exit, cells become (see Glossary, Box 1). Epiblast cells in contact with the responsive to mechanical stimuli. In particular, cell stretching leads to cytotrophoblast differentiate to form an extra-embryonic tissue, an increase in the intracellular levels of calcium, which activate stress the amnion, whereas epiblast cells opposite the cytotrophoblast and metabolic pathways (Verstreken et al., 2019). remain pluripotent (Shahbazi and Zernicka-Goetz, 2018). As a Altogether, these studies provide a glimpse of the complex result, whereas mouse embryos acquire a cylindrical shape, human interplay between transcriptional responses, mechanics, geometry embryos display a disc-shaped morphology. and signalling in pluripotent cells. However, functional studies in the human embryo are needed to determine whether these mechanisms Pluripotency and epiblast shape are conserved from ESCs to embryos and from mice to humans. Epiblast cells at the blastocyst stage display naïve pluripotency, characterised by global hypomethylation, expression of transcription Amnion differentiation factors promoting the naïve state and two active X-chromosomes in A key event in the development of implanting human embryos is the female cells (Nichols and Smith, 2012; Theunissen et al., 2016). This split of epiblast cells into the pluripotent epiblast disc and the developmental ‘blank state’ is lost as embryos implant: genes that differentiated extra-embryonic amniotic epithelium. Our knowledge promote the naïve state are downregulated, whereas post-implantation of the mechanisms that govern this event is very rudimentary, as in factors become upregulated, methylation levels increase and one of most animal models amnion formation takes place at gastrulation the X chromosomes is inactivated in female cells (Nakamura et al., (Dobreva et al., 2010). Much of what we know is derived from 2016; Xiang et al., 2019; Kalkan and Smith, 2014). Importantly, exit studies in monkey embryos (Hill, 1932). Upon implantation, from the naïve pluripotent state happens concomitantly with the epiblast cells in contact with the cytotrophoblast adopt a morphological transformation of the epiblast into an epithelial tissue squamous epithelial morphology and downregulate pluripotency that lines the amniotic cavity, but whether these two events are factors such as NANOG and SOX2, whereas epiblast cells in mechanistically linked was unknown until recently. Using 3D contact with the hypoblast form a columnar epithelium and remain cultures of mouse and human ESCs, and mouse and human pluripotent (Luckett, 1975; Sasaki et al., 2016). This fate split could embryo cultures, it has been shown that amniotic cavity formation be regulated by a gradient of BMP and/or WNT activity, as the is transcriptionally controlled by a pluripotent state transition monkey cytotrophoblast and amnion are a source of WNT and BMP (Shahbazi et al., 2017). Naïve pluripotent cells polarise in response ligands, whereas the hypoblast secretes WNT and BMP inhibitors to the underlying ECM, but apical vesicle exocytosis is compromised. (Sasaki et al., 2016). In agreement with this notion, stimulation of Consequently, failure to dismantle the naïve state leads to impaired human ESCs with BMP leads to the formation of amnion-like cells amniotic cavitation and epiblast epithelialisation both in mouse and (Guo et al., 2020; Minn et al., 2020), BMP signalling is required for human embryos (Shahbazi et al., 2017). In the mouse epiblast, the amnion specification in mouse embryos (Dobreva et al., 2018) and transcription factors Oct4 and Otx2 induce the expression of culture of human ESCs in a soft 3D matrix generates amnion-like podocalyxin, which participates in lumen opening by promoting squamous spheroids in a BMP-dependent manner (Shao et al., electrostatic membrane repulsion of apposing membranes during 2016). Mechanical cues may cooperate with BMP signalling, as vesicle exocytosis. This transcriptional regulation may not be plating human ESCs on a hard surface prevents amniogenesis (Shao conserved in human embryos, as OTX2 is weakly expressed in the et al., 2016). Amnion formation is also regulated by cell density; human epiblast (Xiang et al., 2019), and OCT4 has different functions high cell densities preserve pluripotency and inhibit amniogenesis, in mouse and human blastocysts (Fogarty et al., 2017). Therefore, the whereas low cell densities promote amnion specification (Shao transcription factors that control amniotic cavity formation in human et al., 2017). However, the exact mechanism of amnion embryos remain to be determined. Notwithstanding the specific specification awaits further investigation. mechanism, this is a very clear example of how a cell identity change is necessary for a change in tissue shape. Gastrulation: from pluripotency loss to lineage specification The fate-shape relationship is bidirectional, as changes in cell The day 14 rule limits human embryo research to the first 14 days of shape and mechanics also control pluripotent cell identity. development (Pera, 2017) and, thus, to the period prior to the DEVELOPMENT

6 REVIEW Development (2020) 147, dev190629. doi:10.1242/dev.190629

Morphogenesis: Cell fate: tissue shape changes gene expression changes

Endocytosis Naive pluripotency exit Membrane tension Post-implantation Increased factors FGF activity? Naive genes RNA


Blastocyst Blastocyst Exocytosis Exocytosis and Transcription luminal proteins factors


Epiblast disc

Fig. 3. Proposed cell fate-tissue shape crosstalk during human implantation development. Upon implantation, epiblast cells exit from the naïve pluripotent state and initiate the expression of post-implantation factors. Studies in mouse ESCs have shown that this is controlled by a decrease in membrane tension, which promotes endocytosis and, as a consequence, increased fibroblast growth factor (FGF) signalling activity, which is required for naïve pluripotency exit. Therefore, membrane tension affects the pluripotent state of a cell. Whether this pathway is active in human embryos remains to be explored. Upon naïve pluripotency exit, genes involved in exocytosis become expressed and initiate the process of lumenogenesis to form the amniotic cavity. Exocytic vesicles provide apical membrane and luminal proteins, which are necessary to build a lumen de novo. The transcription factors involved in this process remain to be identified. Question mark denotes a molecular event that has not beem validated in human embryos. formation of the primitive streak (see Glossary, Box 1). observed in the pre-gastrulating human epiblast (Etoc et al., 2016). Consequently, gastrulation cannot be studied in human embryos. Addition of BMP4 triggers pluripotency exit and lineage Given that it is ‘not birth, marriage or death, but gastrulation which commitment. Despite the homogenous presentation of the is truly the most important time in your life’ (Wolpert and Vicente, morphogen, germ layers become radially organised, with TE and 2015), it is not surprising that multiple groups are putting a great amnion on the outside, followed by endoderm, mesoderm and effort into recreating human embryogenesis using stem cells ectoderm towards the centre of the colony (Warmflash et al., 2014; (Shahbazi et al., 2019; Simunovic and Brivanlou, 2017). During Minn et al., 2020). Mechanistic studies revealed that patterning was gastrulation, cells undergo concomitant changes in shape and fate. a consequence of the diffusion of the BMP4 inhibitor noggin At the site where the primitive streak forms, epiblast cells undergo (NOG) and the epithelial architecture of the colony (Etoc et al., epithelial-to-mesenchymal transition (EMT) (see Glossary, Box 1), 2016). As a result of the epithelial nature of human ESCs, BMP lose their pluripotent features and generate mesoderm (see Glossary, receptors become basally localised and therefore inaccessible to the Box 1) and endoderm (see Glossary, Box 1) (Tam and Behringer, apically presented BMP4. However, cells in the border of the colony 1997). Epiblast progenitors that do not ingress through the streak have a less pronounced polarised phenotype, which allows ligand- become ectoderm (see Glossary, Box 1). The onset of gastrulation receptor interactions and, hence, differentiation of the outermost is regulated by the signalling crosstalk between embryonic and cells. Negative feedback by NOG, which is directly induced by extra-embryonic cells, mediated by the concerted action of BMP, BMP4, is also required to explain the fate patterning (Etoc et al., WNT and activin A/Nodal signals and their inhibitors (Tam and 2016). Interestingly, a recent report has shown that BMP receptors Loebel, 2007). These signalling interactions may be further are basolaterally localised in the early post-implantation mouse modulated by mechanical cues and the physical structure of the epiblast, and this is required for the formation of a robust BMP tissue. Modelling the human embryo using stem cells disentangles signalling gradient (Zhang et al., 2019), indicating tissue geometry this complexity by modulating single parameters at a time (Siggia controls pluripotent cell differentiation both in mice and humans and Warmflash, 2018). (Fig. 4). An alternative mechanistic explanation for the emergence of Self-organisation of human ESCs patterning upon BMP4 stimulation has been proposed by Tewary In human ESC cultures, cell density and colony shape and size can et al. (2017). A reaction-diffusion mechanism is initially responsible be precisely controlled using micropattern technology (Théry and for the generation of a self-organised BMP4 gradient. The Piel, 2009; Bauwens et al., 2008; Nazareth et al., 2013). These subsequent interpretation of that gradient and the patterned fate initial studies led to the conclusion that colony size and composition acquisition is consistent with the positional information model. In affect human ESC identity in the absence of exogenous cues support of this idea, decreasing the concentration of BMP4 is (Peerani et al., 2007). sufficient to preserve fate patterning in micropatterns of smaller size Subsequent experiments applied micropattern technology to the (0.2-0.3 mm). This represents a potential mechanism to adapt to study of tissue patterning. When human ESCs are cultured in changes in embryo size, and hence maintain developmental micropatterns of 0.5 mm, they reach a cell density equivalent to that robustness (Tewary et al., 2017). However, a recent report has DEVELOPMENT

7 REVIEW Development (2020) 147, dev190629. doi:10.1242/dev.190629

Cell fate: Morphogenesis: gene expression changes tissue shape changes Polarized mesoderm marker expression Mesenchymal shape RNA

Brachyury A P Unpolarised Decreased BMP receptors EMT transcription cell-cell adhesion Epiblast disc factors Epiblast disc Mesendoderm Increased BMP specification and WNT signalling


Fig. 4. Proposed cell fate-tissue shape crosstalk during human post-implantation development. BMP and WNT signals secreted by the amnion and the trophectoderm lead to expression of mesoderm markers, such as the transcription factor brachyury, in the posterior epiblast. Brachyury induces expression of epithelial-to-mesenchymal transition (EMT) transcription factors, which cause a decrease in the levels of E-cadherin. As a consequence, cell-cell adhesions weaken and cells lose their epithelial morphology and their polarised organisation, such as the polarised localisation of BMP receptors. The unpolarised BMP receptors are able to interact with BMP ligands, increasing BMP signalling activity and inducing mesendoderm specification. proposed that BMP4 does not act as a classical morphogen. In very hydrogels revealed that gastrulation-like domains emerge in areas of small colonies (one to eight cells), the response to BMP4 is binary: high tension due to the release of β-catenin from cell-cell adhesion cells adopt either a pluripotent or a TE fate (Nemashkalo et al., sites and its nuclear translocation, both in mouse and human ESCs 2017). The appearance of mesendodermal fates in a primitive (Muncie et al., 2020; Blin et al., 2018). To dissect the role of streak-like region additionally requires WNT and Nodal activation mechanics during development, future experimental strategies need (Siggia and Warmflash, 2018). to: be based on the use of robust, reproducible and relevant stem cell Self-organised patterns also emerge upon stimulation with WNT models of the embryo (Huch et al., 2017; Van Den Brink et al., (Martyn et al., 2018), which leads to the appearance of an outer 2020); allow mapping of intrinsic stresses and application of mesendoderm ring while inner cells remain pluripotent. Formation extrinsic forces in a precise manner (Vianello and Lutolf, 2019); and of this fate pattern requires E-cadherin cell-cell adhesions, which are use appropriate readouts to assess changes in patterning and more prominent in the colony centre, and dampen WNT activity by morphogenesis. sequestering β-catenin. In addition, the WNT inhibitor DKK1 is expressed in response to WNT and controls the size of the Embryonic-extra-embryonic crosstalk mesendoderm ring (Martyn et al., 2019). In agreement with these We can add an extra layer of complexity to self-organising models micropattern studies, a group of cells in the primitive streak of of human ESCs by investigating how extra-embryonic cells mouse and rabbit embryos expresses both activators and inhibitors modulate human embryonic stem cell fate and shape at of the BMP and WNT signalling pathways (Peng et al., 2016; gastrulation. This is particularly important given the different Idkowiak et al., 2004), indicating there are epiblast-intrinsic organisation of extra-embryonic tissues in mouse and human mechanisms that regulate the position of the primitive streak. embryos. Microfluidics has been recently used to model the These examples demonstrate how simplified 2D models of the epiblast-amnion fate split (Zheng et al., 2019). This led to the embryo can help us dissect the chemical control of tissue patterning; characterisation of the amnion as a crucial signalling centre. but mechanical cues also play a role in patterning. For example, Amniotic epithelial-like cells express high levels of BMP4 and mechanical strains during gastrulation lead to mesoderm induce gastrulation-like events in a WNT-dependent manner in specification via induction of the transcription factor brachyury, pluripotent human ESCs and specification of primordial - and this mechanism may be conserved from flies to humans (Brunet like cells (see Glossary, Box 1) (Zheng et al., 2019). Therefore, et al., 2013). Although the 2D self-organisation of human ESCs whereas in mouse embryos the extra-embryonic ectoderm is the key described so far does not recapitulate the mechanical features of the driver of gastrulation (Tam and Behringer, 1997), in human developing embryo, recent technological advances provide an embryos this role may be played by the amnion. Conversely, the opportunity to explore the contribution of mechanics (Vianello and function of the hypoblast as an inhibitor of BMP and WNT signalling Lutolf, 2019). The use of hydrogels led to the observation that seems to be conserved in mouse, monkey and humans (Xiang et al., matrix stiffness, degradability and composition influence 2019; Stower and Srinivas, 2014; Sasaki et al., 2016). Hypoblast cells proliferation, tissue structure and differentiation in the neural secrete BMP and WNT inhibitors in a polarised manner, and lineage (Ranga et al., 2016; Sun et al., 2014). Similarly, mesoderm therefore the combined action of TE/amnion and hypoblast leads to and endoderm differentiation are enhanced in soft substrates but the generation of a gradient of BMP and WNT activity across the inhibited in stiffer ones (Przybyla et al., 2016; Chen et al., 2020). epiblast. Future studies are needed to dissect the specific

Combining micropattern technology with embryo-like compliant contributions of the cytotrophoblast, hypoblast and amnion in DEVELOPMENT

8 REVIEW Development (2020) 147, dev190629. doi:10.1242/dev.190629 patterning the human epiblast. This could be achieved by combining embryonic and extra-embryonic cells? What is the fate of aneuploid studies on in vitro cultured human embryos (Xiang et al., 2019) with cells in mosaic embryos? Healthy babies can potentially be born in vivo and in vitro cultured monkey embryos (Nakamura et al., 2016; from mosaic aneuploid human embryos (Greco et al., 2015), Niu et al., 2019; Ma et al., 2019) and the development of new stem suggesting aneuploid cells may be selectively eliminated during cell models of the human embryo comprising both embryonic and development. Could this selective elimination be a consequence of extra-embryonic stem cells, as previously used in the mouse the impaired response of aneuploid cells to physical and/or (Harrison et al., 2017; Sozen et al., 2018; Rivron et al., 2018). biochemical cues? And given that 25-50% of spontaneous miscarriages do not show chromosomal abnormalities, what is the When things go wrong: aneuploidy and pregnancy loss reason behind their failed development? Could alterations in the cell Understanding the basic mechanisms of human development is fate-tissue shape crosstalk be responsible for some of these early fundamental to tackling an important clinical problem: the pregnancy losses? Addressing these questions and understanding remarkable inefficiency of human reproduction. It is estimated the mechanisms that lead to pregnancy loss is a first step towards the that only 30-40% of conceptions progress to live birth (Larsen et al., future development of therapeutic interventions. 2013). Development may fail at any time between fertilisation and term, but the early stages are especially crucial. Epidemiological Conclusions studies estimate that 40-50% of embryos are lost before implantation Despite remarkable advances in the field of human development, until (Wilcox et al., 2020). Once the embryo implants, the ongoing very recently human embryo research was mainly descriptive and, as pregnancy can be detected based on the increased levels of human Wilhelm Roux already realised by the end of the 19th century, to chorionic gonadotropin (hCG) (Wilcox et al., 1999). Based on hCG comprehend the mechanisms of development, we must interfere with serum levels, 20-30% of implanted embryos fail to progress beyond it. Today, we are ideally suited to tackle this challenge and decipher week 6 (Wilcox et al., 1988). Owing to the lack of ultrasound how form and function emerge during human embryogenesis. Recent verification, these cases are termed biochemical losses (Larsen et al., evidence indicates that the crosstalk between cell fate and tissue shape 2013; Macklon et al., 2002). The reasons behind this high rate of early plays an important role during key human developmental events, pregnancy loss remain poorly understood. Multiple potential including compaction and polarisation in pre-implantation embryos, mechanisms have been involved, including both alterations in embryonic epithelialisation at the time of implantation and germ embryo development and maternal causes, but further research is lineage commitment at gastrulation. We are just beginning to needed to understand their contribution. understand how cell fate specification events modulate tissue The observation that 50-75% of spontaneous abortions present organisation and, vice versa, how changes in tissue shape modulate karyotypic abnormalities and morphological malformations cell identity and tissue patterning, leading to the emergence of self- (Philipp et al., 2003; Boué et al., 1975) has resulted in a focus on organisation. A number of crucial questions still remain to be the genetic causes of pregnancy loss. While the incidence of meiotic addressed. What are the mechanisms that remodel the implanting aneuploidy in most species is remarkably low, 10-30% of fertilised human embryo? How do physical and chemical cues coordinate human eggs are aneuploid due to female meiotic errors (Hassold morphogenesis and patterning? What signals trigger cell fate and Hunt, 2001; Nagaoka et al., 2012). These chromosome specification during gastrulation? And how are all these events segregation errors are more prevalent in young women and in affected when chromosomal alterations are present? To address these women of advanced age, resulting from whole-chromosome questions, we now possess an adequate set of experimental tools: nondisjunction and premature sister chromatid separation, genome editing has already been successfully used to study gene respectively (Gruhn et al., 2019). Why, when and how embryos function in human embryos (Fogarty et al., 2017); novel imaging harbouring specific aneuploidies fail during embryogenesis remains techniques allow us to map cellular behaviours with unprecedented unknown. All single chromosome aneuploidies can potentially form resolution (McDole et al., 2018); new culture methods have extended blastocysts, but they do so at a lower rate and with a higher incidence the window of human development amenable to investigation of morphological abnormalities. Aneuploid embryos are more (Shahbazi et al., 2016; Deglincerti et al., 2016; Xiang et al., 2019); likely to arrest during EGA, to be developmentally delayed and to and human ESC-embryo chimeras could be potentially used as a gold present a low-quality TE and/or ICM (i.e. few cells and cellular standard to study (De Los Angeles et al., 2015; fragmentation indicative of cell death) (Minasi et al., 2016; Fragouli Alexandrova et al., 2016; Tachibana et al., 2012; Chen et al., 2015), et al., 2013). Moreover, despite forming morphologically normal although stringent criteria to assess lineage contribution and ethical blastocysts, aneuploid embryos display transcriptional and considerations need to be applied (Posfai et al., 2020). In addition, epigenetic alternations that may compromise their subsequent human ESCs can mimic different aspects of embryo development in development (McCallie et al., 2016; Licciardi et al., 2018; Starostik vitro (Shahbazi et al., 2019). These stem cell models of the embryo are et al., 2020). Autosomal monosomies typically lead to biochemical allowing researchers to deconstruct the complexity of human losses, whereas autosomal trisomies are associated with silent development and to decipher the basic principles of self- miscarriage (presence of extra-embryonic membranes but lack of organisation. Last and importantly, the overall increase in assisted embryonic structures) and first-trimester miscarriage (Ljunger et al., reproductive technology cycles over the years, together with the 2005; Martinez et al., 2010; Ferro et al., 2003; Stephenson et al., improvement in embryo selection procedures, leads to the generation 2002). In addition, alterations in the number of chromosomes can be of vast numbers of surplus human embryos (Kushnir et al., 2017), a consequence of mitotic alterations during the first three cleavage which represent an invaluable resource for research. Using this new divisions, which lead to the formation of mosaic human embryos set of tools, we can start to unravel the mysteries of our own (Popovic et al., 2019; Starostik et al., 2020; Mantikou et al., 2012). development. Given the inaccessibility of human embryos at these stages, several fundamental questions remain to be addressed. How do specific Acknowledgements alterations in the number of chromosomes impact on morphogenesis I am grateful to Eric Siggia, Meng Zhu, Bailey Weatherbee, Kirsty Mackinlay and or cell fate specification? Is the response to aneuploidy different in Michele Petruzzelli for critical reading of the manuscript. DEVELOPMENT

9 REVIEW Development (2020) 147, dev190629. doi:10.1242/dev.190629

Competing interests Chen, Y., Niu, Y., Li, Y., Ai, Z., Kang, Y., Shi, H., Xiang, Z., Yang, Z., Tan, T., Si, W. The authors declare no competing or financial interests. et al. (2015). Generation of Cynomolgus Monkey Chimeric Fetuses using Embryonic Stem Cells. Cell Stem Cell 17, 116-124. doi:10.1016/j.stem.2015.06. Funding 004 Chen, Y.-F., Li, Y.-S. J., Chou, C.-H., Chiew, M. Y., Huang, H.-D., Ho, J. H.-C., Work in M.N.S.’s group is funded by the Medical Research Council (MC_UP_1201/ Chien, S. and Lee, O. K. (2020). Control of matrix stiffness promotes endoderm 24) and the European Molecular Biology Organization. Deposited in PMC for lineage specification by regulating SMAD2/3 via lncRNA LINC00458. Sci. Adv., 6. immediate release. Christodoulou, N., Kyprianou, C., Weberling, A., Wang, R., Cui, G., Peng, G., Jing, N. and Zernicka-Goetz, M. (2018). Sequential formation and resolution of References multiple rosettes drive embryo remodelling after implantation. Nat Cell Biol 20, Alarcon, V. B. (2010). Cell polarity regulator PARD6B is essential for trophectoderm 1278-1289. doi:10.1038/s41556-018-0211-3 formation in the preimplantation mouse embryo. Biol Reprod 83, 347-358. doi:10. Coste, B., Mathur, J., Schmidt, M., Earley, T. J., Ranade, S., Petrus, M. J., Dubin, 1095/biolreprod.110.084400 A. E. and Patapoutian, A. (2010). Piezo1 and Piezo2 are essential components Alexandrova, S., Kalkan, T., Humphreys, P., Riddell, A., Scognamiglio, R., of distinct mechanically activated cation channels. Science 330, 55-60. doi:10. Trumpp, A. and Nichols, J. (2016). Selection and dynamics of embryonic stem 1126/science.1193270 cell integration into early mouse embryos. Development 143, 24-34. doi:10.1242/ De Belly, H., Jones, P. H., Paluch, E. K. and Chalut, K. J. (2019). Membrane dev.124602 tension mediated mechanotransduction drives fate choice in embryonic stem Alikani, M. (2005). Epithelial cadherin distribution in abnormal human pre- cells. bioRxiv. implantation embryos. Hum Reprod 20, 3369-3375. doi:10.1093/humrep/dei242 De Los Angeles, A., Ferrari, F., Xi, R., Fujiwara, Y., Benvenisty, N., Deng, H., Amita, M., Adachi, K., Alexenko, A. P., Sinha, S., Schust, D. J., Schulz, L. C., Hochedlinger, K., Jaenisch, R., Lee, S., Leitch, H. G. et al. (2015). Hallmarks of Roberts, R. M. and Ezashi, T. (2013). Complete and unidirectional conversion of pluripotency. Nature 525, 469-478. doi:10.1038/nature15515 human embryonic stem cells to trophoblast by BMP4. Proc Natl Acad Sci U S A De Paepe, C., Cauffman, G., Verloes, A., Sterckx, J., Devroey, P., Tournaye, H., 110, E1212-E1221. doi:10.1073/pnas.1303094110 Liebaers, I. and Van De Velde, H. (2013). Human trophectoderm cells are not yet Anani, S., Bhat, S., Honma-Yamanaka, N., Krawchuk, D. and Yamanaka, Y. committed. Hum Reprod 28, 740-749. doi:10.1093/humrep/des432 (2014). Initiation of Hippo signaling is linked to polarity rather than to cell position in Deglincerti, A., Croft, G. F., Pietila, L. N., Zernicka-Goetz, M., Siggia, E. D. and the pre-implantation mouse embryo. Development 141, 2813-2824. doi:10.1242/ Brivanlou, A. H. (2016). Self-organization of the in vitro attached human embryo. dev.107276 Nature 533, 251-254. doi:10.1038/nature17948 Bauwens, C. L., Peerani, R., Niebruegge, S., Woodhouse, K. A., Kumacheva, E., Desprat, N., Supatto, W., Pouille, P. A., Beaurepaire, E. and Farge, E. (2008). Husain, M. and Zandstra, P. W. (2008). Control of human embryonic stem cell Tissue deformation modulates twist expression to determine anterior midgut colony and aggregate size heterogeneity influences differentiation trajectories. differentiation in Drosophila embryos. Dev Cell 15, 470-477. doi:10.1016/j.devcel. Stem Cells 26, 2300-2310. doi:10.1634/stemcells.2008-0183 2008.07.009 Bedzhov, I. and Zernicka-Goetz, M. (2014). Self-organizing properties of mouse Di Stefano, B., Ueda, M., Sabri, S., Brumbaugh, J., Huebner, A. J., Sahakyan, pluripotent cells initiate morphogenesis upon implantation. Cell 156, 1032-1044. A., Clement, K., Clowers, K. J., Erickson, A. R., Shioda, K. et al. (2018). doi:10.1016/j.cell.2014.01.023 Reduced MEK inhibition preserves genomic stability in naive human embryonic Benham-Pyle, B. W., Pruitt, B. L. and Nelson, W. J. (2015). Cell adhesion. stem cells. Nat Methods 15, 732-740. doi:10.1038/s41592-018-0104-1 Mechanical strain induces E-cadherin-dependent Yap1 and beta-catenin Dobreva, M. P., Pereira, P. N., Deprest, J. and Zwijsen, A. (2010). On the origin of activation to drive cell cycle entry. Science 348, 1024-1027. amniotic stem cells: of mice and men. Int J Dev Biol 54, 761-777. doi:10.1387/ijdb. Bergert, M. L., Lembo, S., Milovanovic, D., Bormel, M., Neveu, P. and Diz- 092935md Muñoz, A. (2019). Cell surface mechanics gate stem cell differentiation. bioRxiv. Dobreva, M. P., Abon Escalona, V., Lawson, K. A., Sanchez, M. N., Ponomarev, Bernardo, A. S., Faial, T., Gardner, L., Niakan, K. K., Ortmann, D., Senner, C. E., L. C., Pereira, P. N. G., Stryjewska, A., Criem, N., Huylebroeck, D., Chuva De Callery, E. M., Trotter, M. W., Hemberger, M., Smith, J. C. et al. (2011). Sousa Lopes, S. M. et al. (2018). Amniotic ectoderm expansion in mouse occurs Brachyury and CDX2 mediate BMP-induced differentiation of human and mouse via distinct modes and requires SMAD5-mediated signalling. Development, 145. pluripotent stem cells into embryonic and extraembryonic lineages. Cell Stem Cell Dong, C., Beltcheva, M., Gontarz, P., Zhang, B., Popli, P., Fischer, L. A., Khan, S. A., Park, K. M., Yoon, E. J., Xing, X. et al. (2020). Derivation of trophoblast 9, 144-155. doi:10.1016/j.stem.2011.06.015 stem cells from naive human pluripotent stem cells. Elife, 9. Blakeley, P., Fogarty, N. M., Del Valle, I., Wamaitha, S. E., Hu, T. X., Elder, K., Dumortier, J. G., Le Verge-Serandour, M., Tortorelli, A. F., Mielke, A., De Plater, Snell, P., Christie, L., Robson, P. and Niakan, K. K. (2015). Defining the three L., Turlier, H. and Maitre, J. L. (2019). Hydraulic fracturing and active coarsening cell lineages of the human blastocyst by single-cell RNA-seq. Development 142, position the lumen of the mouse blastocyst. Science 365, 465-468. doi:10.1126/ 3151-3165. doi:10.1242/dev.123547 science.aaw7709 Blin, G., Wisniewski, D., Picart, C., Thery, M., Puceat, M. and Lowell, S. (2018). Dupont, S., Morsut, L., Aragona, M., Enzo, E., Giulitti, S., Cordenonsi, M., Geometrical confinement controls the asymmetric patterning of brachyury in Zanconato, F., Le Digabel, J., Forcato, M., Bicciato, S. et al. (2011). Role of cultures of pluripotent cells. Development, 145. YAP/TAZ in mechanotransduction. Nature 474, 179-183. doi:10.1038/ Boroviak, T. and Nichols, J. (2017). Primate embryogenesis predicts the hallmarks nature10137 of human naive pluripotency. Development 144, 175-186. doi:10.1242/dev. Durdu, S., Iskar, M., Revenu, C., Schieber, N., Kunze, A., Bork, P., Schwab, Y. 145177 and Gilmour, D. (2014). Luminal signalling links cell communication to tissue ́ Boue, J., Bou, A. and Lazar, P. (1975). Retrospective and prospective architecture during . Nature 515, 120-124. doi:10.1038/ epidemiological studies of 1500 karyotyped spontaneous human abortions. nature13852 Teratology 12, 11-26. doi:10.1002/tera.1420120103 Edwards, R. G., Bavister, B. D. and Steptoe, P. C. (1969). Early stages of Braude, P., Bolton, V. and Moore, S. (1988). Human gene expression first occurs fertilization in vitro of human oocytes matured in vitro. Nature 221, 632-635. between the four- and eight-cell stages of preimplantation development. Nature doi:10.1038/221632a0 332, 459-461. doi:10.1038/332459a0 Edwards, R. G., Steptoe, P. C. and Purdy, J. M. (1970). Fertilization and cleavage Brunet, T., Bouclet, A., Ahmadi, P., Mitrossilis, D., Driquez, B., Brunet, A.-C., in vitro of preovulator human oocytes. Nature 227, 1307-1309. doi:10.1038/ ́ ́ Henry, L., Serman, F., Bealle, G., Menager, C. et al. (2013). Evolutionary 2271307a0 conservation of early mesoderm specification by mechanotransduction in Etoc, F., Metzger, J., Ruzo, A., Kirst, C., Yoney, A., Ozair, M. Z., Brivanlou, A. H. Bilateria. Nat Commun 4, 2821. doi:10.1038/ncomms3821 and Siggia, E. D. (2016). A Balance between Secreted Inhibitors and Edge Bryant, D. M. and Mostov, K. E. (2008). From cells to organs: building polarized Sensing Controls Gastruloid Self-Organization. Dev Cell 39, 302-315. doi:10. tissue. Nat Rev Mol Cell Biol 9, 887-901. doi:10.1038/nrm2523 1016/j.devcel.2016.09.016 Bryant, D. M., Roignot, J., Datta, A., Overeem, A. W., Kim, M., Yu, W., Peng, X., Ferro, J., Martinez, M. C., Lara, C., Pellicer, A., Remohi, J. and Serra, V. (2003). Eastburn, D. J., Ewald, A. J., Werb, Z. et al. (2014). A molecular switch for the Improved accuracy of hysteroembryoscopic biopsies for karyotyping early missed orientation of epithelial cell polarization. Dev Cell 31, 171-187. doi:10.1016/j. abortions. Fertil Steril 80, 1260-1264. doi:10.1016/S0015-0282(03)02195-2 devcel.2014.08.027 Fierro-Gonzalez, J. C., White, M. D., Silva, J. C. and Plachta, N. (2013). Cadherin- Chan, C. J. and Hiiragi, T. (2020). Integration of luminal pressure and signalling in dependent filopodia control preimplantation embryo compaction. Nat Cell Biol 15, tissue self-organization. Development, 147. 1424-1433. doi:10.1038/ncb2875 Chan, C. J., Heisenberg, C. P. and Hiiragi, T. (2017). Coordination of Fogarty, N. M. E., Mccarthy, A., Snijders, K. E., Powell, B. E., Kubikova, N., Morphogenesis and Cell-Fate Specification in Development. Curr Biol 27, Blakeley, P., Lea, R., Elder, K., Wamaitha, S. E., Kim, D. et al. (2017). Genome R1024-R1035. doi:10.1016/j.cub.2017.07.010 editing reveals a role for OCT4 in human embryogenesis. Nature 550, 67-73. Chan, C. J., Costanzo, M., Ruiz-Herrero, T., Monke, G., Petrie, R. J., Bergert, M., doi:10.1038/nature24033 Diz-Munoz, A., Mahadevan, L. and Hiiragi, T. (2019). Hydraulic control of Fragouli, E., Alfarawati, S., Spath, K., Jaroudi, S., Sarasa, J., Enciso, M. and mammalian embryo size and cell fate. Nature 571, 112-116. doi:10.1038/s41586- Wells, D. (2013). The origin and impact of embryonic aneuploidy. Hum Genet 132,

019-1309-x 1001-1013. doi:10.1007/s00439-013-1309-0 DEVELOPMENT

10 REVIEW Development (2020) 147, dev190629. doi:10.1242/dev.190629

Gardner, R. L. (1983). Origin and differentiation of extraembryonic tissues in the Krendl, C., Shaposhnikov, D., Rishko, V., Ori, C., Ziegenhain, C., Sass, S., mouse. Int Rev Exp Pathol 24, 63-133. Simon, L., Muller, N. S., Straub, T., Brooks, K. E. et al. (2017). GATA2/3- Gasser, R. F., Cork, R. J., Stillwell, B. J. and Mcwilliams, D. T. (2014). Rebirth of TFAP2A/C transcription factor network couples human pluripotent stem cell human embryology. Dev Dyn 243, 621-628. doi:10.1002/dvdy.24110 differentiation to trophectoderm with repression of pluripotency. Proc Natl Acad Goehring, N. W., Trong, P. K., Bois, J. S., Chowdhury, D., Nicola, E. M., Hyman, Sci U S A 114, E9579-E9588. doi:10.1073/pnas.1708341114 A. A. and Grill, S. W. (2011). Polarization of PAR proteins by advective triggering Kushnir, V. A., Barad, D. H., Albertini, D. F., Darmon, S. K. and Gleicher, N. of a pattern-forming system. Science 334, 1137-1141. doi:10.1126/science. (2017). Systematic review of worldwide trends in assisted reproductive technology 1208619 2004-2013. Reprod Biol Endocrinol 15, 6. doi:10.1186/s12958-016-0225-2 Greco, E., Minasi, M. G. and Fiorentino, F. (2015). Healthy Babies after Larsen, E. C., Christiansen, O. B., Kolte, A. M. and Macklon, N. (2013). New Intrauterine Transfer of Mosaic Aneuploid Blastocysts. N Engl J Med 373, insights into mechanisms behind miscarriage. BMC Med 11, 154. doi:10.1186/ 2089-2090. doi:10.1056/NEJMc1500421 1741-7015-11-154 Green, J. B. and Sharpe, J. (2015). Positional information and reaction-diffusion: Leung, C. Y. and Zernicka-Goetz, M. (2013). Angiomotin prevents pluripotent two big ideas in combine. Development 142, 1203-1211. lineage differentiation in mouse embryos via Hippo pathway-dependent and doi:10.1242/dev.114991 -independent mechanisms. Nat Commun 4, 2251. doi:10.1038/ncomms3251 Gruhn, J. R., Zielinska, A. P., Shukla, V., Blanshard, R., Capalbo, A., Leung, C. Y., Zhu, M. and Zernicka-Goetz, M. (2016). Polarity in Cell-Fate Cimadomo, D., Nikiforov, D., Chan, A. C., Newnham, L. J., Vogel, I. et al. Acquisition in the Early Mouse Embryo. Curr Top Dev Biol 120, 203-234. doi:10. (2019). Chromosome errors in human eggs shape natural fertility over 1016/bs.ctdb.2016.04.008 reproductive life span. Science 365, 1466-1469. doi:10.1126/science.aav7321 Li, S., Edgar, D., Fassler, R., Wadsworth, W. and Yurchenco, P. D. (2003). The Guo, G., Stirparo, G. G., Strawbridge, S., Yang, J., Clarke, J., Li, M. A., Myers, S., role of laminin in embryonic cell polarization and tissue organization. Dev Cell 4, Ozel, B. N., Nichols, J. and Smith, A. (2020). Trophectoderm potency is retained 613-624. doi:10.1016/S1534-5807(03)00128-X exclusively in human naive cells. bioRxiv. Liang, X., Zhou, Q., Li, X., Sun, Y., Lu, M., Dalton, N., Ross, Jr., J. and Chen, J. Hamidi, S., Nakaya, Y., Nagai, H., Alev, C., Kasukawa, T., Chhabra, S., Lee, R., (2005). PINCH1 plays an essential role in early murine Niwa, H., Warmflash, A., Shibata, T. et al. (2020). Mesenchymal-epithelial but is dispensable in ventricular cardiomyocytes. Mol Cell Biol 25, 3056-3062. transition regulates initiation of pluripotency exit before gastrulation. doi:10.1128/MCB.25.8.3056-3062.2005 Development, 147. Licciardi, F., Lhakhang, T., Kramer, Y. G., Zhang, Y., Heguy, A. and Tsirigos, A. Hannezo, E. and Heisenberg, C.-P. (2019). Mechanochemical Feedback Loops in (2018). Human blastocysts of normal and abnormal karyotypes display distinct Development and Disease. Cell 178, 12-25. doi:10.1016/j.cell.2019.05.052 transcriptome profiles. Sci Rep 8, 14906. doi:10.1038/s41598-018-33279-0 Harrison, S. E., Sozen, B., Christodoulou, N., Kyprianou, C. and Zernicka- Lindenberg, S., Nielsen, M. H. and Lenz, S. (1985). In vitro studies of human Goetz, M. (2017). Assembly of embryonic and extraembryonic stem cells to mimic blastocyst implantation. Ann N Y Acad Sci 442, 368-374. doi:10.1111/j.1749- embryogenesis in vitro. Science, 356. 6632.1985.tb37541.x Hassold, T. and Hunt, P. (2001). To err (meiotically) is human: the genesis of Linneberg-Agerholm, M., Wong, Y. F., Romero Herrera, J. A., Monteiro, R. S., human aneuploidy. Nat Rev Genet 2, 280-291. doi:10.1038/35066065 Anderson, K. G. V. and Brickman, J. M. (2019). Naive human pluripotent stem Heisenberg, C. P. and Bellaiche, Y. (2013). Forces in tissue morphogenesis and cells respond to Wnt, Nodal and LIF signalling to produce expandable naive extra- embryonic endoderm. Development, 146. patterning. Cell 153, 948-962. doi:10.1016/j.cell.2013.05.008 Liu, J., He, X., Corbett, S. A., Lowry, S. F., Graham, A. M., Fassler, R. and Li, S. Hertig, A. T., Rock, J. and Adams, E. C. (1956). A description of 34 human ova (2009). Integrins are required for the differentiation of visceral endoderm. J Cell Sci within the first 17 days of development. Am J Anat 98, 435-493. doi:10.1002/aja. 122, 233-242. doi:10.1242/jcs.037663 1000980306 Ljunger, E., Cnattingius, S., Lundin, C. and Annerén, G. (2005). Chromosomal Hill, J. P. (1932). The developmental history of the primates. Philos Trans R Soc anomalies in first-trimester miscarriages. Acta Obstet Gynecol Scand 84, Lond B Biol Sci 221, 45-178. doi:10.1098/rstb.1932.0002 1103-1107. doi:10.1111/j.0001-6349.2005.00882.x Hirate, Y., Hirahara, S., Inoue, K., Suzuki, A., Alarcon, V. B., Akimoto, K., Hirai, Luckett, W. P. (1975). The development of primordial and definitive amniotic T., Hara, T., Adachi, M., Chida, K. et al. (2013). Polarity-dependent distribution of cavities in early Rhesus monkey and human embryos. Am J Anat 144, 149-167. angiomotin localizes Hippo signaling in preimplantation embryos. Curr Biol 23, doi:10.1002/aja.1001440204 1181-1194. doi:10.1016/j.cub.2013.05.014 Ma, H., Zhai, J., Wan, H., Jiang, X., Wang, X., Wang, L., Xiang, Y., He, X., Zhao, Home, P., Saha, B., Ray, S., Dutta, D., Gunewardena, S., Yoo, B., Pal, A., Vivian, Z. A., Zhao, B. et al. (2019). In vitro culture of cynomolgus monkey embryos J. L., Larson, M., Petroff, M. et al. (2012). Altered subcellular localization of beyond early gastrulation. Science, 366. transcription factor TEAD4 regulates first mammalian cell lineage commitment. Macklon, N. S., Geraedts, J. P. and Fauser, B. C. (2002). Conception to ongoing Proc Natl Acad Sci U S A 109, 7362-7367. doi:10.1073/pnas.1201595109 pregnancy: the ‘black box’ of early pregnancy loss. Hum Reprod Update 8, Horii, M., Bui, T., Touma, O., Cho, H. Y. and Parast, M. M. (2019). An Improved 333-343. doi:10.1093/humupd/8.4.333 Two-Step Protocol for Trophoblast Differentiation of Human Pluripotent Stem Maitre, J. L., Niwayama, R., Turlier, H., Nedelec, F. and Hiiragi, T. (2015). Cells. Curr Protoc Stem Cell Biol 50, e96. doi:10.1002/cpsc.96 Pulsatile cell-autonomous contractility drives compaction in the mouse embryo. Huch, M., Knoblich, J. A., Lutolf, M. P. and Martinez-Arias, A. (2017). The hope Nat Cell Biol 17, 849-855. doi:10.1038/ncb3185 and the hype of organoid research. Development 144, 938-941. doi:10.1242/dev. Maitre, J. L., Turlier, H., Illukkumbura, R., Eismann, B., Niwayama, R., Nedelec, 150201 F. and Hiiragi, T. (2016). Asymmetric division of contractile domains couples cell Idkowiak, J., Weisheit, G., Plitzner, J. and Viebahn, C. (2004). Hypoblast controls positioning and fate specification. Nature 536, 344-348. doi:10.1038/nature18958 mesoderm generation and axial patterning in the gastrulating rabbit embryo. Dev Mantikou, E., Wong, K. M., Repping, S. and Mastenbroek, S. (2012). Molecular Genes Evol 214, 591-605. doi:10.1007/s00427-004-0436-y origin of mitotic aneuploidies in preimplantation embryos. Biochim Biophys Acta Ivec, M., Kovacic, B. and Vlaisavljevic, V. (2011). Prediction of human blastocyst 1822, 1921-1930. doi:10.1016/j.bbadis.2012.06.013 development from morulas with delayed and/or incomplete compaction. Fertil. Marcos, J., Perez-Albala, S., Mifsud, A., Molla, M., Landeras, J. and Meseguer, Steril., 96, 1473-1478e2. M. (2015). Collapse of blastocysts is strongly related to lower implantation Iwata, K., Yumoto, K., Sugishima, M., Mizoguchi, C., Kai, Y., Iba, Y. and Mio, Y. success: a time-lapse study. Hum Reprod 30, 2501-2508. doi:10.1093/humrep/ (2014). Analysis of compaction initiation in human embryos by using time-lapse dev216 cinematography. J Assist Reprod Genet 31, 421-426. doi:10.1007/s10815-014- Martinez, M. C., Mendez, C., Ferro, J., Nicolas, M., Serra, V. and Landeras, J. 0195-2 (2010). Cytogenetic analysis of early nonviable pregnancies after assisted Kalkan, T. and Smith, A. (2014). Mapping the route from naive pluripotency to reproduction treatment. Fertil Steril 93, 289-292. doi:10.1016/j.fertnstert.2009.07. lineage specification. Philos. Trans. R. Soc. Lond. B Biol. Sci., 369. 989 Keibel, F. and Mall, F. P. (1912). Manual of Human Embryology. JB Lippincott Martyn, I., Kanno, T. Y., Ruzo, A., Siggia, E. D. and Brivanlou, A. H. (2018). Self- Company. organization of a human organizer by combined Wnt and Nodal signalling. Nature Kim, J., Kim, S. H. and Jun, J. H. (2017). Prediction of blastocyst development and 558, 132-135. doi:10.1038/s41586-018-0150-y implantation potential in utero based on the third cleavage and compaction times Martyn, I., Brivanlou, A. H. and Siggia, E. D. (2019). A wave of WNT signaling in mouse pre-implantation embryos. J Reprod Dev 63, 117-125. doi:10.1262/jrd. balanced by secreted inhibitors controls primitive streak formation in micropattern 2016-129 colonies of human embryonic stem cells. Development, 146. Kirby, T. J. and Lammerding, J. (2018). Emerging views of the nucleus as a cellular Mccallie, B. R., Parks, J. C., Patton, A. L., Griffin, D. K., Schoolcraft, W. B. and mechanosensor. Nat Cell Biol 20, 373-381. doi:10.1038/s41556-018-0038-y Katz-Jaffe, M. G. (2016). Hypomethylation and Genetic Instability in Monosomy Kojima, Y., Tam, O. H. and Tam, P. P. (2014). Timing of developmental events in Blastocysts May Contribute to Decreased Implantation Potential. PLoS One 11, the early mouse embryo. Semin Cell Dev Biol 34, 65-75. doi:10.1016/j.semcdb. e0159507. doi:10.1371/journal.pone.0159507 2014.06.010 Mcdole, K., Guignard, L., Amat, F., Berger, A., Malandain, G., Royer, L. A., Korotkevich, E., Niwayama, R., Courtois, A., Friese, S., Berger, N., Buchholz, F. Turaga, S. C., Branson, K. and Keller, P. J. (2018). In Toto Imaging and and Hiiragi, T. (2017). The Apical Domain Is Required and Sufficient for the First Reconstruction of Post-Implantation Mouse Development at the Single-Cell Level.

Lineage Segregation in the Mouse Embryo. Dev. Cell, 40, 235-247e7. Cell, 175, 859-876e33. DEVELOPMENT

11 REVIEW Development (2020) 147, dev190629. doi:10.1242/dev.190629

Meng, Y., Cai, K. Q., Moore, R., Tao, W., Tse, J. D., Smith, E. R. and Xu, X.-X. in the pathogenesis of developmental defects of early failed pregnancies. Hum (2017). Pten facilitates epiblast epithelial polarization and proamniotic lumen Reprod 18, 1724-1732. doi:10.1093/humrep/deg309 formation in early mouse embryos. Dev Dyn 246, 517-530. doi:10.1002/dvdy. Pieters, T. and Van Roy, F. (2014). Role of cell-cell adhesion complexes in 24503 embryonic stem cell biology. J Cell Sci 127, 2603-2613. doi:10.1242/jcs.146720 Minasi, M. G., Colasante, A., Riccio, T., Ruberti, A., Casciani, V., Scarselli, F., Plusa, B., Frankenberg, S., Chalmers, A., Hadjantonakis, A. K., Moore, C. A., Spinella, F., Fiorentino, F., Varricchio, M. T. and Greco, E. (2016). Correlation Papalopulu, N., Papaioannou, V. E., Glover, D. M. and Zernicka-Goetz, M. between aneuploidy, standard morphology evaluation and morphokinetic (2005). Downregulation of Par3 and aPKC function directs cells towards the Icm in development in 1730 biopsied blastocysts: a consecutive case series study. the preimplantation mouse embryo. J Cell Sci 118, 505-515. doi:10.1242/jcs. Hum Reprod 31, 2245-2254. doi:10.1093/humrep/dew183 01666 Minn, K. T., Fu, Y. C., He, S., George, S. C., Anastasio, M. A., Morris, S. A. and Popovic, M., Dhaenens, L., Taelman, J., Dheedene, A., Bialecka, M., De Sutter, Solnica-Krezel, L. (2020). High-resolution transcriptional and morphogenetic P., Chuva De Sousa Lopes, S. M., Menten, B. and Heindryckx, B. (2019). profiling of cells from micropatterned human embryonic stem cell gastruloid Extended in vitro culture of human embryos demonstrates the complex nature of cultures. bioRxiv. diagnosing chromosomal mosaicism from a single trophectoderm biopsy. Hum. Mischler, A., Karakis, V., Mahinthakumar, J., Carberry, C., San Miguel, A., Reprod. Rager, J., Fry, R. and Rao, B. M. (2019). Two distinct trophectoderm lineage Posfai, E., Petropoulos, S., De Barros, F. R. O., Schell, J. P., Jurisica, I., stem cells from human pluripotent stem cells. bioRxiv. Sandberg, R., Lanner, F. and Rossant, J. (2017). Position- and Hippo signaling- Mizobe, Y., Ezono, Y., Tokunaga, M., Oya, N., Iwakiri, R., Yoshida, N., Sato, Y., dependent plasticity during lineage segregation in the early mouse embryo. Elife, 6. Onoue, N. and Miyoshi, K. (2017). Selection of human blastocysts with a high Posfai, E., Schell, J. P., Janiszewski, A., Rovic, I., Murray, A., Bradshaw, B., implantation potential based on timely compaction. J Assist Reprod Genet 34, Pardon, T., El Bakkali, M., Talon, I., De Geest, N. et al. (2020). Defining 991-997. doi:10.1007/s10815-017-0962-y totipotency using criteria of increasing stringency. bioRxiv. Muncie, J. M., Ayad, N. M. E., Lakins, J. N. and Weaver, V. M. (2020). Mechanics Przybyla, L., Lakins, J. N. and Weaver, V. M. (2016). Tissue Mechanics regulate human embryonic stem cell self-organization to specify mesoderm. Orchestrate Wnt-Dependent Human Embryonic Stem Cell Differentiation. Cell bioRxiv. Stem Cell 19, 462-475. doi:10.1016/j.stem.2016.06.018 Munne, S., Tomkin, G. and Cohen, J. (2009). Selection of embryos by morphology Qin, H., Hejna, M., Liu, Y., Percharde, M., Wossidlo, M., Blouin, L., Durruthy- is less effective than by a combination of aneuploidy testing and morphology Durruthy, J., Wong, P., Qi, Z., Yu, J. et al. (2016). YAP Induces Human Naive observations. Fertil Steril 91, 943-945. doi:10.1016/j.fertnstert.2007.06.082 Pluripotency. Cell Rep 14, 2301-2312. doi:10.1016/j.celrep.2016.02.036 Nagaoka, S. I., Hassold, T. J. and Hunt, P. A. (2012). Human aneuploidy: Ralston, A., Cox, B. J., Nishioka, N., Sasaki, H., Chea, E., Rugg-Gunn, P., Guo, mechanisms and new insights into an age-old problem. Nat Rev Genet 13, G., Robson, P., Draper, J. S. and Rossant, J. (2010). Gata3 regulates 493-504. doi:10.1038/nrg3245 trophoblast development downstream of Tead4 and in parallel to Cdx2. Nakamura, T., Okamoto, I., Sasaki, K., Yabuta, Y., Iwatani, C., Tsuchiya, H., Development 137, 395-403. doi:10.1242/dev.038828 Seita, Y., Nakamura, S., Yamamoto, T. and Saitou, M. (2016). A developmental Ranga, A., Gobaa, S., Okawa, Y., Mosiewicz, K., Negro, A. and Lutolf, M. P. coordinate of pluripotency among mice, monkeys and humans. Nature 537, (2014). 3D niche microarrays for systems-level analyses of cell fate. Nat Commun 57-62. doi:10.1038/nature19096 5, 4324. doi:10.1038/ncomms5324 Nazareth, E. J., Ostblom, J. E., Lucker, P. B., Shukla, S., Alvarez, M. M., Oh, Ranga, A., Girgin, M., Meinhardt, A., Eberle, D., Caiazzo, M., Tanaka, E. M. and S. K., Yin, T. and Zandstra, P. W. (2013). High-throughput fingerprinting of Lutolf, M. P. (2016). Neural tube morphogenesis in synthetic 3D human pluripotent stem cell fate responses and lineage bias. Nat Methods 10, microenvironments. Proc Natl Acad Sci U S A 113, E6831-E6839. doi:10.1073/ 1225-1231. doi:10.1038/nmeth.2684 pnas.1603529113 Nemashkalo, A., Ruzo, A., Heemskerk, I. and Warmflash, A. (2017). Morphogen Rayon, T., Menchero, S., Nieto, A., Xenopoulos, P., Crespo, M., Cockburn, K., and community effects determine cell fates in response to BMP4 signaling in Canon, S., Sasaki, H., Hadjantonakis, A. K., De La Pompa, J. L. et al. (2014). human embryonic stem cells. Development 144, 3042-3053. doi:10.1242/dev. Notch and hippo converge on Cdx2 to specify the trophectoderm lineage in the 153239 mouse blastocyst. Dev Cell 30, 410-422. doi:10.1016/j.devcel.2014.06.019 Niakan, K. K. and Eggan, K. (2013). Analysis of human embryos from zygote to Rivron, N. C., Frias-Aldeguer, J., Vrij, E.-J., Boisset, J. C., Korving, J., Vivié, J., blastocyst reveals distinct gene expression patterns relative to the mouse. Dev Truckenmüller, R. K., Van Oudenaarden, A., Van Blitterswijk, C. A. and Biol 375, 54-64. doi:10.1016/j.ydbio.2012.12.008 Geijsen, N. (2018). Blastocyst-like structures generated solely from stem cells. Nichols, J. and Gardner, R. L. (1984). Heterogeneous differentiation of external Nature 557, 106-111. doi:10.1038/s41586-018-0051-0 cells in individual isolated early mouse inner cell masses in culture. J Embryol Exp Rock, J. and Menkin, M. F. (1944). In Vitro Fertilization and Cleavage of Human Morphol 80, 225-240. Nichols, J. and Smith, A. (2012). Pluripotency in the embryo and in culture. Cold Ovarian Eggs. Science 100, 105-107. doi:10.1126/science.100.2588.105 Spring Harb Perspect Biol 4, a008128. doi:10.1101/cshperspect.a008128 Ryan, A. Q., Chan, C. J., Graner, F. and Hiiragi, T. (2019). Lumen Expansion Nikas, G., Ao, A., Winston, R. M. and Handyside, A. H. (1996). Compaction and Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse surface polarity in the human embryo in vitro. Biol Reprod 55, 32-37. doi:10.1095/ Blastocyst Formation. Dev. Cell, 51, 684-697e4. biolreprod55.1.32 Saiz, N., Grabarek, J. B., Sabherwal, N., Papalopulu, N. and Plusa, B. (2013). Nishimura, H., Takano, K., Tanimura, T. and Yasuda, M. (1968). Normal and Atypical protein kinase C couples cell sorting with primitive endoderm maturation abnormal development of human embryos: first report of the analysis of 1213 in the mouse blastocyst. Development 140, 4311-4322. doi:10.1242/dev.093922 intact embryos. Teratology 1, 281-290. doi:10.1002/tera.1420010306 Sakai, T., Li, S., Docheva, D., Grashoff, C., Sakai, K., Kostka, G., Braun, A., Niu, Y., Sun, N., Li, C., Lei, Y., Huang, Z., Wu, J., Si, C., Dai, X., Liu, C., Wei, J. Pfeifer, A., Yurchenco, P. D. and Fassler, R. (2003). Integrin-linked kinase (ILK) et al. (2019). Dissecting primate early post-implantation development using long- is required for polarizing the epiblast, cell adhesion, and controlling actin term in vitro embryo culture. Science, 366. accumulation. Genes Dev 17, 926-940. doi:10.1101/gad.255603 Niwa, H., Toyooka, Y., Shimosato, D., Strumpf, D., Takahashi, K., Yagi, R. and Samarage, C. R., White, M. D., Alvarez, Y. D., Fierro-Gonzalez, J. C., Henon, Y., Rossant, J. (2005). Interaction between Oct3/4 and Cdx2 determines Jesudason, E. C., Bissiere, S., Fouras, A. and Plachta, N. (2015). Cortical trophectoderm differentiation. Cell 123, 917-929. doi:10.1016/j.cell.2005.08.040 Tension Allocates the First Inner Cells of the Mammalian Embryo. Dev Cell 34, Noli, L., Capalbo, A., Ogilvie, C., Khalaf, Y. and Ilic, D. (2015). Discordant Growth 435-447. doi:10.1016/j.devcel.2015.07.004 of Monozygotic Twins Starts at the Blastocyst Stage: A Case Study. Stem Cell Sasaki, H. (2015). Position- and polarity-dependent Hippo signaling regulates cell Reports 5, 946-953. doi:10.1016/j.stemcr.2015.10.006 fates in preimplantation mouse embryos. Semin Cell Dev Biol 47-48, 80-87. Peerani, R., Rao, B. M., Bauwens, C., Yin, T., Wood, G. A., Nagy, A., Kumacheva, doi:10.1016/j.semcdb.2015.05.003 E. and Zandstra, P. W. (2007). Niche-mediated control of human embryonic stem Sasaki, K., Nakamura, T., Okamoto, I., Yabuta, Y., Iwatani, C., Tsuchiya, H., cell self-renewal and differentiation. Embo J 26, 4744-4755. doi:10.1038/sj.emboj. Seita, Y., Nakamura, S., Shiraki, N., Takakuwa, T. et al. (2016). The Germ Cell 7601896 Fate of Cynomolgus Monkeys Is Specified in the Nascent Amnion. Dev Cell 39, Peng, G., Suo, S., Chen, J., Chen, W., Liu, C., Yu, F., Wang, R., Chen, S., Sun, N., 169-185. doi:10.1016/j.devcel.2016.09.007 Cui, G. et al. (2016). Spatial Transcriptome for the Molecular Annotation of Schwartz, M. A. (2010). Integrins and extracellular matrix in mechanotransduction. Lineage Fates and Cell Identity in Mid-gastrula Mouse Embryo. Dev Cell 36, Cold Spring Harb Perspect Biol 2, a005066. doi:10.1101/cshperspect.a005066 681-697. doi:10.1016/j.devcel.2016.02.020 Shahbazi, M. N. and Zernicka-Goetz, M. (2018). Deconstructing and Pera, M. F. (2017). Human embryo research and the 14-day rule. Development 144, reconstructing the mouse and human early embryo. Nat Cell Biol 20, 878-887. 1923-1925. doi:10.1242/dev.151191 doi:10.1038/s41556-018-0144-x Petropoulos, S., Edsgard, D., Reinius, B., Deng, Q., Panula, S. P., Codeluppi, S., Shahbazi, M. N., Jedrusik, A., Vuoristo, S., Recher, G., Hupalowska, A., Bolton, Plaza Reyes, A., Linnarsson, S., Sandberg, R. and Lanner, F. (2016). Single- V., Fogarty, N. M., Campbell, A., Devito, L. G., Ilic, D. et al. (2016). Self- Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human organization of the human embryo in the absence of maternal tissues. Nat Cell Preimplantation Embryos. Cell, 165, 1012-1026. Biol 18, 700-708. doi:10.1038/ncb3347 Philipp, T., Philipp, K., Reiner, A., Beer, F. and Kalousek, D. K. (2003). Shahbazi, M. N., Scialdone, A., Skorupska, N., Weberling, A., Recher, G., Zhu,

Embryoscopic and cytogenetic analysis of 233 missed abortions: factors involved M., Jedrusik, A., Devito, L. G., Noli, L., Macaulay, I. C. et al. (2017). Pluripotent DEVELOPMENT

12 REVIEW Development (2020) 147, dev190629. doi:10.1242/dev.190629

state transitions coordinate morphogenesis in mouse and human embryos. Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, Nature 552, 239-243. doi:10.1038/nature24675 J. J., Marshall, V. S. and Jones, J. M. (1998). Embryonic stem cell lines derived Shahbazi, M. N., Siggia, E. D. and Zernicka-Goetz, M. (2019). Self-organization of from human blastocysts. Science 282, 1145-1147. doi:10.1126/science.282. stem cells into embryos: A window on early mammalian development. Science 5391.1145 364, 948-951. doi:10.1126/science.aax0164 Turing, A. M. (1952). The chemical basis of morphogenesis. Philos Trans R Soc Shao, Y., Taniguchi, K., Gurdziel, K., Townshend, R. F., Xue, X., Yong, K. M., Lond B Biol Sci 237, 37-72. doi:10.1098/rstb.1952.0012 Sang, J., Spence, J. R., Gumucio, D. L. and Fu, J. (2016). Self-organized Van Den Brink, S. C., Alemany, A., Van Batenburg, V., Moris, N., Blotenburg, M., amniogenesis by human pluripotent stem cells in a biomimetic implantation-like Vivie, J., Baillie-Johnson, P., Nichols, J., Sonnen, K. F., Martinez Arias, A. niche. Nat. Mater. et al. (2020). Single-cell and spatial transcriptomics reveal somitogenesis in Shao, Y., Taniguchi, K., Townshend, R. F., Miki, T., Gumucio, D. L. and Fu, J. gastruloids. Nature. (2017). A pluripotent stem cell-based model for post-implantation human amniotic Vanneste, E., Voet, T., Le Caignec, C., Ampe, M., Konings, P., Melotte, C., sac development. Nat Commun 8, 208. doi:10.1038/s41467-017-00236-w Debrock, S., Amyere, M., Vikkula, M., Schuit, F. et al. (2009). Chromosome Sheng, G. (2015). Epiblast morphogenesis before gastrulation. Dev Biol 401, 17-24. instability is common in human cleavage-stage embryos. Nat Med 15, 577-583. doi:10.1016/j.ydbio.2014.10.003 doi:10.1038/nm.1924 Shettles, L. B. (1955). A morula stage of human ovum developed in vitro. Fertil Steril Verstreken, C. M., Labouesse, C., Agley, C. C. and Chalut, K. J. (2019). 6, 287-289. doi:10.1016/S0015-0282(16)32040-4 Embryonic stem cells become mechanoresponsive upon exit from ground state of Siggia, E. D. and Warmflash, A. (2018). Modeling Mammalian Gastrulation With pluripotency. Open Biol 9, 180203. doi:10.1098/rsob.180203 Embryonic Stem Cells. Curr Top Dev Biol 129, 1-23. doi:10.1016/bs.ctdb.2018. Vianello, S. and Lutolf, M. P. (2019). Understanding the Mechanobiology of Early 03.001 Mammalian Development through Bioengineered Models. Dev Cell 48, 751-763. Simunovic, M. and Brivanlou, A. H. (2017). Embryoids, organoids and gastruloids: doi:10.1016/j.devcel.2019.02.024 new approaches to understanding embryogenesis. Development 144, 976-985. Warmflash, A., Sorre, B., Etoc, F., Siggia, E. D. and Brivanlou, A. H. (2014). A doi:10.1242/dev.143529 method to recapitulate early embryonic spatial patterning in human embryonic Skiadas, C. C., Jackson, K. V. and Racowsky, C. (2006). Early compaction on day stem cells. Nat Methods 11, 847-854. doi:10.1038/nmeth.3016 3 may be associated with increased implantation potential. Fertil Steril 86, Weimar, C. H., Post Uiterweer, E. D., Teklenburg, G., Heijnen, C. J. and 1386-1391. doi:10.1016/j.fertnstert.2006.03.051 Macklon, N. S. (2013). In-vitro model systems for the study of human embryo- Smyth, N., Vatansever, H. S., Murray, P., Meyer, M., Frie, C., Paulsson, M. and interactions. Reprod Biomed Online 27, 461-476. doi:10.1016/j. Edgar, D. (1999). Absence of basement membranes after targeting the LAMC1 rbmo.2013.08.002 gene results in embryonic lethality due to failure of endoderm differentiation. J Cell West, R. C., Ming, H., Logsdon, D. M., Sun, J., Rajput, S. K., Kile, R. A., Biol 144, 151-160. doi:10.1083/jcb.144.1.151 Schoolcraft, W. B., Roberts, R. M., Krisher, R. L., Jiang, Z. et al. (2019). Sozen, B., Amadei, G., Cox, A., Wang, R., Na, E., Czukiewska, S., Chappell, L., Dynamics of trophoblast differentiation in peri-implantation-stage human Voet, T., Michel, G., Jing, N. et al. (2018). Gastrulating complete embryo-like embryos. Proc Natl Acad Sci U S A 116, 22635-22644. doi:10.1073/pnas. structures reconstituted from stem cells in vitro. Nat. Cell Biol. 1911362116 Starostik, M. R., Sosina, O. A. and Mccoy, R. (2020). Single-cell analysis of human Wigger, M., Kisielewska, K., Filimonow, K., Plusa, B., Maleszewski, M. and embryos reveals diverse patterns of aneuploidy and mosaicism. bioRxiv. Suwinska, A. (2017). Plasticity of the inner cell mass in mouse blastocyst is Stephenson, M. D., Awartani, K. A. and Robinson, W. P. (2002). Cytogenetic restricted by the activity of FGF/MAPK pathway. Sci Rep 7, 15136. doi:10.1038/ analysis of miscarriages from couples with recurrent miscarriage: a case-control s41598-017-15427-0 study. Hum Reprod 17, 446-451. doi:10.1093/humrep/17.2.446 Wilcox, A. J., Weinberg, C. R., O’connor, J. F., Baird, D. D., Schlatterer, J. P., Steptoe, P. C., Edwards, R. G. and Purdy, J. M. (1971). Human blastocysts grown Canfield, R. E., Armstrong, E. G. and Nisula, B. C. (1988). Incidence of early in culture. Nature 229, 132-133. doi:10.1038/229132a0 loss of pregnancy. N Engl J Med 319, 189-194. doi:10.1056/ Stirparo, G. G., Boroviak, T., Guo, G., Nichols, J., Smith, A. and Bertone, P. NEJM198807283190401 (2018). Integrated analysis of single-cell embryo data yields a unified Wilcox, A. J., Baird, D. D. and Weinberg, C. R. (1999). Time of implantation of the transcriptome signature for the human pre-implantation epiblast. Development, conceptus and loss of pregnancy. N Engl J Med 340, 1796-1799. doi:10.1056/ 145. NEJM199906103402304 Stower, M. J. and Srinivas, S. (2014). Heading forwards: anterior visceral Wilcox, A. J., Harmon, Q., Doody, K., Wolf, D. P. and Adashi, E. Y. (2020). endoderm migration in patterning the mouse embryo. Philos. Trans. R. Soc. Lond. Preimplantation loss of fertilized human ova: estimating the unobservable. Hum. B Biol. Sci., 369. Reprod. Strumpf, D., Mao, C. A., Yamanaka, Y., Ralston, A., Chawengsaksophak, K., Wolpert, L. (1969). Positional information and the spatial pattern of cellular Beck, F. and Rossant, J. (2005). Cdx2 is required for correct cell fate differentiation. J Theor Biol 25, 1-47. doi:10.1016/S0022-5193(69)80016-0 specification and differentiation of trophectoderm in the mouse blastocyst. Wolpert, L. and Vicente, C. (2015). An interview with Lewis Wolpert. Development Development 132, 2093-2102. doi:10.1242/dev.01801 142, 2547-2548. doi:10.1242/dev.127373 Sun, Y., Yong, K. M., Villa-Diaz, L. G., Zhang, X., Chen, W., Philson, R., Weng, S., Wong, C. C., Loewke, K. E., Bossert, N. L., Behr, B., De Jonge, C. J., Baer, T. M. Xu, H., Krebsbach, P. H. and Fu, J. (2014). Hippo/YAP-mediated rigidity- and Reijo Pera, R. A. (2010). Non-invasive imaging of human embryos before dependent motor neuron differentiation of human pluripotent stem cells. Nat Mater embryonic genome activation predicts development to the blastocyst stage. Nat 13, 599-604. doi:10.1038/nmat3945 Biotechnol 28, 1115-1121. doi:10.1038/nbt.1686 Tachibana, M., Sparman, M., Ramsey, C., Ma, H., Lee, H. S., Penedo, M. C. and Xiang, L., Yin, Y., Zheng, Y., Ma, Y., Li, Y., Zhao, Z., Guo, J., Ai, Z., Niu, Y., Duan, Mitalipov, S. (2012). Generation of chimeric rhesus monkeys. Cell 148, 285-295. K. et al. (2019). A developmental landscape of 3D-cultured human pre- doi:10.1016/j.cell.2011.12.007 gastrulation embryos. Nature. Takashima, Y., Guo, G., Loos, R., Nichols, J., Ficz, G., Krueger, F., Oxley, D., Xu, R. H., Chen, X., Li, D. S., Li, R., Addicks, G. C., Glennon, C., Zwaka, T. P. and Santos, F., Clarke, J., Mansfield, W. et al. (2014). Resetting Transcription Factor Thomson, J. A. (2002). BMP4 initiates human embryonic stem cell differentiation Control Circuitry toward Ground-State Pluripotency in Human. Cell 158, to trophoblast. Nat Biotechnol 20, 1261-1264. doi:10.1038/nbt761 1254-1269. doi:10.1016/j.cell.2014.08.029 Yamada,S.,Hill,M.andTakakuwa,T.(2015). Human embryology. In New Tam, P. P. and Behringer, R. R. (1997). Mouse gastrulation: the formation of a Discoveries in Embryology (ed. B. Wu), pp. 97-124. IntechOpen. mammalian body plan. Mech Dev 68, 3-25. doi:10.1016/S0925-4773(97)00123-8 Yan, L., Yang, M., Guo, H., Yang, L., Wu, J., Li, R., Liu, P., Lian, Y., Zheng, X., Tam, P. P. and Loebel, D. A. (2007). Gene function in mouse embryogenesis: get Yan, J. et al. (2013). Single-cell RNA-Seq profiling of human preimplantation set for gastrulation. Nat Rev Genet 8, 368-381. doi:10.1038/nrg2084 embryos and embryonic stem cells. Nat Struct Mol Biol 20, 1131-1139. doi:10. Taniguchi, K., Shao, Y., Townshend, R. F., Tsai, Y. H., Delong, C. J., Lopez, 1038/nsmb.2660 S. A., Gayen, S., Freddo, A. M., Chue, D. J., Thomas, D. J. et al. (2015). Lumen Zhang, Z., Zwick, S., Loew, E., Grimley, J. S. and Ramanathan, S. (2019). Mouse Formation Is an Intrinsic Property of Isolated Human Pluripotent Stem Cells. Stem embryo geometry drives formation of robust signaling gradients through receptor Cell Reports 5, 954-962. doi:10.1016/j.stemcr.2015.10.015 localization. Nat. Commun. 10, 4516. doi:10.1038/s41467-019-12533-7 Tewary, M., Ostblom, J., Prochazka, L., Zulueta-Coarasa, T., Shakiba, N., Zheng, Y., Xue, X., Shao, Y., Wang, S., Esfahani, S. N., Li, Z., Muncie, J. M., Fernandez-Gonzalez, R. and Zandstra, P. W. (2017). A stepwise model of Lakins, J. N., Weaver, V. M., Gumucio, D. L. et al. (2019). Controlled modelling Reaction-Diffusion and Positional-Information governs self-organized human of human epiblast and amnion development using stem cells. Nature 573, peri-gastrulation-like patterning. Development. 421-425. doi:10.1038/s41586-019-1535-2 Théry, M. and Piel, M. (2009). Adhesive micropatterns for cells: a microcontact Zhou, F., Wang, R., Yuan, P., Ren, Y., Mao, Y., Li, R., Lian, Y., Li, J., Wen, L., Yan, printing protocol. Cold Spring Harb Protoc, doi:10.1101/pdb.prot5255 L. et al. (2019). Reconstituting the transcriptome and Dna methylome landscapes Theunissen, T. W., Friedli, M., He, Y., Planet, E., O’neil, R. C., Markoulaki, S., of human implantation. Nature 572, 660-664. doi:10.1038/s41586-019-1500-0 Pontis, J., Wang, H., Iouranova, A., Imbeault, M. et al. (2016). Molecular Criteria Zhu, M., Leung, C. Y., Shahbazi, M. N. and Zernicka-Goetz, M. (2017). for Defining the Naive Human Pluripotent State. Cell Stem Cell 19, 502-515. Actomyosin polarisation through PLC-PKC triggers symmetry breaking of the

doi:10.1016/j.stem.2016.06.011 mouse embryo. Nat Commun 8, 921. doi:10.1038/s41467-017-00977-8 DEVELOPMENT

13 REVIEW Development (2020) 147, dev190629. doi:10.1242/dev.190629

Zhu, P., Guo, H., Ren, Y., Hou, Y., Dong, J., Li, R., Lian, Y., Fan, X., Hu, B., Gao, Y. Zhu, M., Wang, P., Handford, C. E., Na, J. and Zernicka-Goetz, M. (2020). et al. (2018). Single-cell DNA methylome sequencing of human preimplantation Transcriptional control of apical protein clustering drives de novo cell polarity embryos. Nat Genet 50, 12-19. doi:10.1038/s41588-017-0007-6 establishment in the early mouse embryo. bioRxiv. DEVELOPMENT