REVIEW

Mechanisms of human embryo development: from cell fateto tissue shape and backMarta N. Shahbazi*

ABSTRACTGene regulatory networks and tissue morphogenetic events drive theemergence of shape and function: the pillars of embryo development.Although model systems offer a window into the molecular biology ofcell fate and tissue shape, mechanistic studies of our owndevelopment have so far been technically and ethically challenging.However, recent technical developments provide the tools todescribe, manipulate and mimic human embryos in a dish, thusopening a new avenue to exploring human development. Here, Idiscuss the evidence that supports a role for the crosstalk betweencell fate and tissue shape during early human embryogenesis. This isa critical developmental period, when the body plan is laid out andmany pregnancies fail. Dissecting the basic mechanisms thatcoordinate cell fate and tissue shape will generate an integratedunderstanding of early embryogenesis and new strategies fortherapeutic intervention in early pregnancy loss.

KEY WORDS: Human embryo, Morphogenesis, Cell fate, Embryonicstem cells, Aneuploidy, Polarisation

IntroductionDevelopmental biologists are faced with a question of form andfunction. Starting from a relatively simple spore, seed or egg,multiple cell types arise that perform specific functions. Thesedifferent cells organise into diverse configurations, leading to theemergence of tissues of a defined form. Development requiresmechanical changes in cell and tissue shape, which drivemorphogenesis, and gene expression changes, which regulate cellfate decisions and tissue patterning. These two developmentalprocesses are not independent, as changes in cell fate can modify thearchitecture of tissues, and vice versa (Chan et al., 2017).Mechanochemical feedback at the molecular, cellular and tissuelevels coordinates this crosstalk and instructs tissue self-organisation(see Glossary, Box 1) (Hannezo and Heisenberg, 2019).Molecular motors generate mechanical forces that are transmitted

across tissues via the cytoskeleton and cell-cell adhesion proteins(Heisenberg and Bellaiche, 2013). In the embryo, this leads tochanges in cell shape and position, to large tissue-scaledeformations, and consequently to morphogenesis. Cells sensemechanical cues – a process termed mechanosensation – viaextracellular matrix (ECM) adhesion proteins (Schwartz, 2010),cell-cell adhesion proteins (Benham-Pyle et al., 2015), stretch-

activated ion channels (Coste et al., 2010), mechanosensitivetranscription factors (Dupont et al., 2011) or directly by the nucleus(Kirby and Lammerding, 2018). Once sensed, mechanical cuesare transduced into biochemical signals – a process known asmechanotransduction (Chan et al., 2017). The conversion ofmechanical cues into biochemical signals leads to changes ingene expression and protein activity that control cell behaviour, cellfate specification and tissue patterning.

Current consensus focuses on two main ideas to explain theemergence of tissue patterns in response tomorphogen (see Glossary,Box 1) signals. In the ‘positional information’ model (Wolpert,1969), the concentration of a morphogen serves as a coordinate of theposition of a cell within a tissue. Cells respond to a specificmorphogen concentration making a specific fate choice. Therefore,an existing asymmetry is transformed into a pattern of cell fates. Onthe contrary, in the reaction-diffusion model (Turing, 1952), patternformation is a self-organising phenomenon. The basic idea behindthis model is the presence of a short-range activator that induces itsown expression and the expression of a long-range inhibitor. Aspontaneous increase in the local concentration of the activator (asymmetry-breaking event) will induce a further increase in the levelsof the activator and expression of the inhibitor. However, as theinhibitor has a higher diffusion rate, this will lead to a peak ofactivation surrounded by a valley of repression. These twomechanisms, positional information and reaction-diffusion, co-existduring development to generate patterns (Green and Sharpe, 2015).

The use of model organisms allows us to explore the mechanismsthat orchestrate the crosstalk between cell fate and tissue shape. Forexample, cytoskeletal forces lead to the acquisition of cell polarityand the differential segregation of cell fate determinants inC. elegans (Goehring et al., 2011), an emerging lumen trapsfibroblast growth factor (FGF) molecules, which lead todifferentiation in the lateral line primordium of zebrafish (Durduet al., 2014), and tissue deformations control midgut differentiationinDrosophila embryos (Desprat et al., 2008). As human beings, ourown development remains the most significant to study, yet is stillmysterious due to technical and ethical limitations (Box 2). In thisReview, I draw upon knowledge gained from studies in modelorganisms, embryonic stem cell research and human embryology topropose mechanistic models of three critical developmental events:compaction and polarisation at the cleavage stage; embryonicepithelialisation at the time of implantation; and pluripotent celldifferentiation at gastrulation (Fig. 1). The emerging picturesupports a role for the crosstalk between tissue shape and cell fateas a determinant of human embryogenesis.

Compaction and polarisation: the first morphogenetictransformationCompactionAt the 8- to 16-cell stage, human embryos undergo the process ofcompaction; blastomeres (see Glossary, Box 1) adhere tightly to

MRC Laboratory of Molecular Biology, Francis Crick Avenue, CambridgeBiomedical Campus, Cambridge CB2 0QH, UK.

*Author for correspondence (mshahbazi@mrc-lmb.cam.ac.uk)

M.N.S., 0000-0002-1599-5747

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (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.

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each other, maximising their cell-cell contact area, which leads theembryo to acquire a compressed morphology. This process isconserved in all mammalian species studied so far, but the timingvaries. The precise timing of the onset and completion ofcompaction is important for the successful development of bothmouse and human embryos (Mizobe et al., 2017; Kim et al., 2017;Skiadas et al., 2006). Human embryos in which compaction is eitherinitiated prematurely or delayed have a lower potential to formblastocysts (see Glossary, Box 1). Premature compaction isassociated with cytokinetic failure and multi-nuclear blastomeres(Iwata et al., 2014), whereas delayed compaction is associated witha reduced number of inner cell mass (ICM) cells (see Glossary,Box 1) (Ivec et al., 2011).

The mechanisms that drive compaction in human embryosremain unknown and, instead, have mainly been explored usingmouse embryos. The key players are the actomyosin cytoskeletonand the cell adhesion protein E-cadherin. The actomyosincytoskeleton undergoes pulsed contractions that generate forceand increase membrane tension. This happens specifically at thecell-free surface of the embryo, as E-cadherin keeps contractilitylow at cell-cell adhesion sites (Maitre et al., 2015). An alternativemodel proposes that E-cadherin-containing filopodia are the majordrivers of compaction. By binding to and promoting elongation of

Box 1. GlossaryAmnion. An extra-embryonic membrane that protects thedeveloping foetus from mechanical damage and the maternalimmune response.Amniotes. A clade of vertebrates in which the embryo develops whilebeing protected by extra-embryonic membranes, including the amnion. Itencompasses reptiles, birds and mammals.Amniotic cavity. Fluid-filled sac located between the amnion and theembryo, the function of which is to protect the embryo from mechanicaldamage and the maternal immune system.Blastocoel. Fluid-filled cavity present in blastocyst stage embryos.Blastocyst. An embryo composed of an outer trophectoderm layer, aninner cell mass and an internal blastocoel cavity.Blastomeres. Cells of the early embryo, formed by cleavage of thezygote.Cytotrophoblast. Trophectoderm stem cell compartment of the humanembryo. It plays a key role during blastocyst implantation and it generatesdifferentiated trophectoderm cells such as syncytiotrophoblast andextravillous trophoblast.Ectoderm. Outermost primary germ layer that gives rise to the nervoussystem, the epidermis and its appendages.Embryonic genome activation. The process whereby zygotictranscription is initiated and takes over the maternally stored mRNAsand proteins. In human embryos, it takes place at the four- to eight-cellstage transition.Embryoid body. Three dimensional aggregates of pluripotent stem cellscommonly used to study differentiation.Endoderm. Innermost primary germ layer that gives rise to theepithelial cells of the gastrointestinal, respiratory, endocrine andurinary systems.Epiblast. The pluripotent embryonic tissue that gives rise to germlineand soma, as well as the extra-embryonic amnion.Epithelial-to-mesenchymal transition. Cellular program that leads tothe loss of epithelial features (i.e. polarised organisation and cell-celladhesion) and the acquisition of a mesenchymal phenotype. It plays akey role during gastrulation.Extra-embryonic ectoderm. Trophectoderm-derived tissue that formsduring early post-implantation in mouse embryos.Inner cell mass. A group of cells located inside the blastocyst that givesrise to the epiblast and the hypoblast.Morphogen. A signalling molecule that determines cell fates in aconcentration-dependent manner.Mesoderm. Primary germ layer positioned in between the ectoderm andthe endoderm. It gives rise to muscle, bone, connective tissue, bloodvessels, red and white blood cells, and the mesenchyme of variousorgans such as skin, kidney or gonads.Primary yolk sac. Extra-embryonic membrane formed by hypoblastcells around 7 to 8 days post-fertilisation. By E12-15, it gives rise to asecondary yolk sac, the primary functions of which are hematopoiesisand nutrient transport.Primitive endoderm (mouse) or hypoblast (human). An extra-embryonic tissue characteristic of pre-implantation blastocysts thatgives rise to the yolk sac.Primitive streak. Transient structure that appears in the posteriorepiblast and marks the start of gastrulation.Primordial germ cells. Specialised cells, precursors of the gametes,that are specified in the epiblast of mammalian embryos during earlypost-implantation development.Pro-amniotic cavity. Luminal cavity that is formed in the mouse epiblastat E5.0. The fusion between the epiblast and extra-embryonic ectodermcavities leads to the formation of the amniotic cavity by E5.75 in mouseembryos.Self-organisation. Spontaneous generation of ordered structures thatresults from the interaction between elements that have no previouspattern.Sialomucins. Glycosylated proteins of the mucin family that contain theacidic sugar sialic acid.Trophectoderm. An extra-embryonic tissue present in pre-implantationembryos that gives rise to the placenta.

Box 2. Historical perspective of human embryodevelopmentThe birth of human embryology as a scientific discipline is intimatelylinked to the creation of human embryo collections (Yamada et al., 2015;Gasser et al., 2014). The pioneering work of Franklin Mall led to thecreation of the Carnegie collection in 1887, which harbours more than10,000 human embryo specimens, and established the basic stagingcriteria for the developmental classification of human embryos (Keibeland Mall, 1912). Other collections were later created, such as the Kyotocollection, which today holds ∼44,000 specimens (Nishimura et al.,1968). Much of our current textbook knowledge of human development isderived from the early descriptive studies of these samples.The development of in vitro fertilisation (IVF) of human eggs initiated a

revolution in human embryo and stem cell research and humanreproduction (Edwards et al., 1969; Rock and Menkin, 1944; Shettles,1955). This initial milestone was followed by the development ofconditions to culture fertilised human eggs in vitro for up to 5-6 days(Edwards et al., 1970; Steptoe et al., 1971), and ultimately led to the birthof the first IVF baby in 1978, thanks to the tireless efforts of RobertEdwards, Patrick Steptoe and Jean Purdy. Since then, the field of humanembryology has flourished. IVF has allowed scientists to describe thedynamics of key morphogenetic processes during early humandevelopment, such as cleavage, compaction and blastulation (Wonget al., 2010; Marcos et al., 2015; Iwata et al., 2014); to characterise celllineage specification events by studying the transcriptional andepigenetic profiles of all the cells present in a developing humanembryo (Niakan and Eggan, 2013; Petropoulos et al., 2016; Braudeet al., 1988; Zhu et al., 2018); to identify genetic and chromosomalabnormalities that compromise human embryo development (Munneet al., 2009; Vanneste et al., 2009); and, perhaps more importantly, toestablish human embryonic stem cell lines (Thomson et al., 1998), whichon their own have revolutionised our approach to studying humandevelopment and devising regenerative therapies. However, untilrecently, gene function could not be studied in the context of humanembryos. The recent generation of OCT4 knockout human embryosrepresents a turning point in the field (Fogarty et al., 2017). This studyhighlighted differences in gene function between mouse and humans,and established a gold standard for functional studies in humanembryos. Thus, human embryology is becoming an experimentalscience; I argue that, in the years to come, we will witness a surge inthe number of mechanistic studies exploring our own development.

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the apical surface of neighbouring blastomeres, they trigger the cellshape changes that take place during compaction in a non-cell-autonomous manner (Fierro-Gonzalez et al., 2013). Whether thesemechanisms are active in human embryos remains unknown. Interms of timing, studies in mouse embryos suggest that the onset ofcompaction could be controlled by the decrease in the nuclear/cytoplasmic ratio that takes place during the early cleavage divisionsand/or the dilution of a potential inhibitor of compaction present inthe zygote (Kojima et al., 2014). These are interesting hypothesesthat require further experimental validation.

PolarisationConcomitant with the process of compaction, cells acquireapicobasal polarity. In mouse embryos, the basolateral domainconcentrates cell adhesion proteins, such as E-cadherin, whereas theapical domain is enriched in apical polarity proteins, such as thepartitioning defective (Par) complex and cytoskeletal components(Leung et al., 2016). Recent studies in mice have revealed that

protein kinase C (PKC) activation at the eight-cell stage triggersactomyosin polarisation via RhoA (Zhu et al., 2017). In addition,transcription is required for Par complex polarisation. Thetranscription factors Tfap2c and Tead4 are expressed afterembryonic genome activation (EGA, see Glossary, Box 1) andinduce the expression of several regulators of the actin cytoskeleton,which become progressively upregulated from the two- to eight-cellstage. This leads to a remodelling of the actin network that isnecessary for the clustering of apical polarity proteins, andculminates with the formation of the mature apical domain. As aresult, the combined artificial activation of RhoA and expression ofTfap2c and Tead4 is sufficient to induce premature embryopolarisation and to advance morphogenesis (Zhu et al., 2020).

Studies in human embryos revealed the presence of apicalmicrovilli and basolateral E-cadherin by the time embryos compact(Alikani, 2005; Nikas et al., 1996), but the exact sequence of eventsand the mechanisms leading to human embryo polarisation remainto be explored. Given that EGA precedes polarisation, it is tempting

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Fig. 1. Overview of human and mouse embryo development. Upon fertilisation, mouse and human embryos undergo a series of cleavage divisions. Theembryonic genome becomes activated by the two-cell stage in mouse embryos and at the four/eight-cell stage transition in human embryos. It is followed bycompaction and polarisation, which occur at the eight-cell stage in mouse embryos, and between the eight- to 16-cell stage in human embryos. Formation of ahollow 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. Theembryonic epiblast loses its naïve pluripotent character, becomes epithelial and forms the pro-amniotic cavity (mouse), which spans both the epiblast and thetrophectoderm-derived extra-embryonic ectoderm, and the amniotic cavity (human). A key difference between mouse and human embryos relates to theformation of the amnion.Whereas in mouse embryos amnion formation takes place during gastrulation, in human embryos a subset of epiblast cells differentiatesto form the squamous amniotic epithelium during early post-implantation (E10). As a result, the human epiblast acquires a disc shape and the mouse epiblast acylindrical 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-derivedtissues are shown in blue and trophectoderm-derived tissues are shown in green.

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to speculate that, as in mouse embryos, EGA controls the timing ofhuman embryo polarisation via as yet unknown transcription factors(Zhu et al., 2020) (Fig. 2).

The first cell fate decisionPolarisation is fundamental for the divergence of embryonic andextra-embryonic lineages in mouse embryos. In support of thisnotion, loss-of-function studies demonstrate that Par complexcomponents are needed for the acquisition of an extra-embryonictrophectoderm (TE) (see Glossary, Box 1) fate (Plusa et al., 2005;Alarcon, 2010). Cells that inherit the apical domain differentiatetowards TE, whereas cells that do not inherit the apical domain andare internalised become the ICM, and hence the precursors of theembryonic epiblast (see Glossary, Box 1) and extra-embryonicprimitive endoderm (PE) (Korotkevich et al., 2017) (see Glossary,Box 1). Inner cell allocation is mainly regulated by apicalconstriction, heterogeneities in cortical tension and, to a lesserextent, by the orientation of cell division (Samarage et al., 2015;Anani et al., 2014). This leads to the first spatial segregation of cellsin the mouse embryo: outer polarised cells and inner apolar cells.Therefore, two morphogenetic cues, polarity and position, regulatethe first cell fate decision in mouse embryos.Mechanistically, the transcription factor Yap is responsible for

the ICM-TE bifurcation in mouse embryos (Sasaki, 2015). Inpolarised cells, Yap translocates to the nucleus and binds thetranscription factor Tead4, which leads to the expression of TEgenes such as Cdx2 (Rayon et al., 2014). In turn, Cdx2 inhibits theexpression of the ICM-specific pluripotency markers Oct4 (Niwaet al., 2005) and Nanog (Strumpf et al., 2005). By the 32-cell stage,Cdx2+ cells are irreversibly committed to the TE fate (Posfai et al.,2017; Nichols and Gardner, 1984; Gardner, 1983). Yap localisationis regulated by several upstream factors. In apolar cells, the Hippopathway component angiomotin activates the Hippo kinase Lats,which phosphorylates Yap, leading to its cytoplasmic sequestration.In polarised cells, angiomotin is recruited to the apical domain andbecomes inactivated, allowing Yap to translocate to the nucleus(Leung and Zernicka-Goetz, 2013; Hirate et al., 2013). Differences

in actomyosin contractility and tension between polar and apolarcells also regulate Yap localisation (Maitre et al., 2016).

Our knowledge of lineage specification in human embryosremains rudimentary. Single-cell sequencing analyses havegenerated a spatiotemporal catalogue of gene expression and haverevealed a number of transcriptional differences between mouse andhuman embryos (Niakan and Eggan, 2013; Blakeley et al., 2015;Petropoulos et al., 2016; Yan et al., 2013). At the compacted morulastage (embryonic day E4), the transcription of TE genes such asGATA3 and PDGFA is initiated, although cells still express ICMgenes (Petropoulos et al., 2016). It is not until the early blastocyststage (embryonic day E5) when lineage-specific markers start tobecome mutually exclusive. Aggregation experiments have shownthat E5 TE cells still retain the ability to form ICM cells (De Paepeet al., 2013), and, conversely, isolated human ICMs can alsogenerate TE cells (Guo et al., 2020). These results indicate that, evenif the ICM-TE lineage segregation takes places at the blastocyststage (Petropoulos et al., 2016), cells are not yet fully committed.

Analysis of protein expression and localisation has alsouncovered interesting differences between mouse and humanembryos. YAP localises to the nucleus both in TE and epiblastcells in human blastocysts (Qin et al., 2016), but becomes restrictedto the TE at the late blastocyst stage (Noli et al., 2015), in agreementwith a delayed ICM-TE segregation in human embryos whencompared with mouse. Supporting a potential role for YAP duringTE specification, loss of YAP prevents human embryonic stem cell(ESC) differentiation to the TE fate (Mischler et al., 2019; Guoet al., 2020). Surprisingly, CDX2, a master TE regulator in mouseembryos, is not expressed until the late blastocyst stage in humanembryos (Niakan and Eggan, 2013); its target genes in the mouse,Eomes and Elf5, are not expressed in pre-implantation human TE(Blakeley et al., 2015); and loss of OCT4 in human embryos leads toa downregulation of CDX2 expression (Fogarty et al., 2017),indicating that, in contrast to mouse embryos, OCT4 and CDX2 donot mutually repress each other in human embryos (Niwa et al.,2005; Ralston et al., 2010). Globally, these findings raise thepossibility that different transcription factors control the

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

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specification of human TE (Fig. 2). GATA3 is an interestingcandidate, as its downregulation in primate embryos inhibits TEdifferentiation (Krendl et al., 2017) and because it is a downstreamtarget of TEAD4/YAP (Home et al., 2012) that inhibits OCT4expression (Krendl et al., 2017) in human ESCs differentiated to TE.Future studies are needed to determine whether YAP and GATA3play a role in TE specification in human embryos. This is especiallyimportant as protocols to differentiate human ESCs into TE may notreflect the in vivo specification route. Differentiation of humanESCs upon addition of BMP4 has been widely used to obtain TE-like cells (Amita et al., 2013; Xu et al., 2002). However, variousreports suggest that cells acquire an extra-embryonic and embryonicmesoderm and/or extra-embryonic amniotic fate rather than a TEfate (Guo et al., 2020; Bernardo et al., 2011). Although newdifferentiation protocols are being developed (Guo et al., 2020;Mischler et al., 2019; Horii et al., 2019; Dong et al., 2020), these donot reflect the 3D organisation of the embryo, and the potentialcontribution of embryo polarisation and cell position to TEspecification cannot be explored. Therefore, the molecular playersinvolved in human TE specification, and whether, as in the mouse,polarisation controls the first lineage segregation in human embryosremain to be determined.

The implanting embryo: a global transformationThe second cell fate decisionHuman and mouse embryos reach the blastocyst stage at embryonicdays E5 and E3.5, respectively. Blastocyst formation involves aprocess of cavitation to form the blastocoel (see Glossary, Box 1). Inmouse embryos, blastocoel formation is driven by the accumulationof pressurised fluid between cells. This fluid moves from the outsidemedium into the intercellular space as a consequence of an osmoticgradient, leading to the formation of microlumens that coarsen toform a single lumen (Dumortier et al., 2019). In turn, luminalpressure leads to increased cortical tension in the TE and tightjunction maturation to allow lumen expansion (Chan et al., 2019).This is a perfect example of how mechanical cues, in this casehydrostatic pressure, affect differentiation (Chan and Hiiragi, 2020).Once the embryo reaches the blastocyst stage, it is ready to

implant in the maternal uterus. At this stage, mouse and humanembryos undergo a second cell fate specification event: the ICMsegregates into pluripotent epiblast and extra-embryonic hypoblast(see Glossary, Box 1). Whereas in mouse embryos the specificationof TE cells precedes the segregation of ICM cells into epiblast andPE (the mouse equivalent of the human hypoblast), the timing ofepiblast-hypoblast segregation in human embryos is still underdebate. Although some studies report a concomitant specification ofthe three lineages, epiblast, hypoblast and TE, in human embryos(Petropoulos et al., 2016), others have identified an early ICM statethat precedes the epiblast-hypoblast split (Stirparo et al., 2018).Upon specification, human hypoblast cells become apicobasallypolarised and express apical proteins such as the sialomucin (seeGlossary, Box 1) protein podocalyxin (Shahbazi et al., 2017). Inmouse embryos, polarisation is required for PE maintenance.Inhibition of atypical protein kinase C (aPKC), a component of thePar complex, or β1-integrin, a cell-ECM adhesion protein, leads todefects in epithelialisation and PE maturation (Saiz et al., 2013; Liuet al., 2009). Studies using human ESCs suggest that aPKC mayalso control the epiblast-hypoblast lineage segregation. Inhibition ofaPKC is needed to maintain naïve pre-implantation-like pluripotenthuman ESCs (Takashima et al., 2014). These cells are competent todifferentiate into extra-embryonic endoderm cells, and this has beenproposed to be dependent on aPKC signalling (Linneberg-

Agerholm et al., 2019), although this hypothesis remains to beformally validated.

A recent study in mouse embryos revealed an unexpected role forthe blastocoel during epiblast-PE specification (Ryan et al., 2019).The authors propose that lumen expansion is needed for correctlineage segregation (Ryan et al., 2019). Interestingly, in isolatedICMs, epiblast and PE progenitors segregate, although with anincreased proportion of PE to epiblast cells (Wigger et al., 2017).This result suggests that, although the second cell fate decision cantake place in the absence of the blastocoel, it may be required to fine-tune the relative numbers of epiblast and PE progenitors.

Human embryo culture beyond implantationOur knowledge of human embryo development at and beyondimplantation (E7) is very scarce. It has been mainly limited to theanalysis of human embryo specimens from the Carnegie collection(Box 2), which have revealed that implanting blastocysts undergoglobal morphological remodelling to form a disc-shaped embryo(Hertig et al., 1956). Conventional human embryo culture methodsonly allow embryo growth and survival up to E6-7 (late blastocyststage). Co-cultures with endometrial cells have traditionally beenused to explore the crosstalk between the embryo and the uterus(Weimar et al., 2013; Lindenberg et al., 1985), but whether theembryo undergoes proper morphogenesis in this setting is unclear.Recently, human embryos have been cultured up to day 13 in vitro,thus permitting insight into the major morphologicaltransformations of post-implantation human development – TEdifferentiation (West et al., 2019), amniotic cavitation (see Glossary,Box 1) and primary yolk sac (see Glossary, Box 1) formation – inthe absence of interactions with maternal tissues (Deglincerti et al.,2016; Shahbazi et al., 2016). This culture method has been takenforward by the addition of a 3D matrix, which allows amnion (seeGlossary, Box 1) specification and further development up to thegastrula stage (Xiang et al., 2019). These systems open the door tostudy human embryo development beyond implantation; e.g. theyhave been used to generate a single-cell transcriptome andmethylome map of post-implantation human embryos (Zhouet al., 2019; Xiang et al., 2019). But given the lack of an in vivoreference, comparisons with primate embryos are important forvalidation (Nakamura et al., 2016; Boroviak and Nichols, 2017; Maet al., 2019; Niu et al., 2019).

Epiblast epithelialisation and amniotic cavitationThe first transformation that epiblast cells undergo is the formationof an epithelial tissue that lines the amniotic cavity. This process isevolutionarily conserved in all amniotes (see Glossary, Box 1) andis a fundamental prerequisite for gastrulation, lineage specificationand developmental progression (Sheng, 2015). Mutations thatimpair pro-amniotic cavity (see Glossary, Box 1) formation lead todevelopmental arrest in mouse embryos and failed differentiation inembryoid bodies (see Glossary, Box 1) (Sakai et al., 2003; Lianget al., 2005; Smyth et al., 1999).

In mouse and human embryos, epiblast epithelialisation takesplace during implantation (Shahbazi and Zernicka-Goetz, 2018). Inresponse to ECM proteins secreted by the extra-embryonic tissues(Li et al., 2003), epiblast cells become polarised and form a rosette-like structure that undergoes lumenogenesis to form the amnioticcavity (Bedzhov and Zernicka-Goetz, 2014; Luckett, 1975). In themouse, epiblast polarisation is triggered by β1-integrin signallingand requires phosphatase and tensin homologue (Pten) and integrin-linked kinase (Ilk) activity (Meng et al., 2017; Sakai et al., 2003),whereas lumenogenesis is triggered by exocytosis of apical vesicles

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and membrane separation (Shahbazi et al., 2017), a process known ashollowing (Bryant and Mostov, 2008). In the human context, furthermechanistic insight has been gained through the use of 3D ESCcultures, which recapitulate the processes of epithelialisation andcavity formation (Shahbazi et al., 2016; Taniguchi et al., 2015).Whereas actin polymerisation promotes cavity formation, actomyosincontractility and the activity of Rho-associated protein kinase (ROCK)prevent lumenogenesis (Taniguchi et al., 2015). Accordingly, theaddition of a ROCK inhibitor to human and monkey embryo culturepromotes amniotic cavity formation (Xiang et al., 2019; Niu et al.,2019). The positive effect of ROCK inhibition during lumenogenesishas been described in other models of hollowing (Bryant et al., 2014),suggesting it is evolutionarily conserved.Once the incipient amniotic cavity is formed, mouse and human

embryos acquire different morphologies (Fig. 1). In the mouseembryo, the TE-derived extra-embryonic ectoderm also undergoes acavitation event. The subsequent fusion of the epiblast and extra-embryonic ectoderm cavities leads to the formation of the pro-amniotic cavity, which spans both tissues (Christodoulou et al.,2018). In contrast, the human TE gives rise to the cytotrophoblast(see Glossary, Box 1). Epiblast cells in contact with thecytotrophoblast differentiate to form an extra-embryonic tissue,the amnion, whereas epiblast cells opposite the cytotrophoblastremain pluripotent (Shahbazi and Zernicka-Goetz, 2018). As aresult, whereas mouse embryos acquire a cylindrical shape, humanembryos display a disc-shaped morphology.

Pluripotency and epiblast shapeEpiblast cells at the blastocyst stage display naïve pluripotency,characterised by global hypomethylation, expression of transcriptionfactors promoting the naïve state and two active X-chromosomes infemale cells (Nichols and Smith, 2012; Theunissen et al., 2016). Thisdevelopmental ‘blank state’ is lost as embryos implant: genes thatpromote the naïve state are downregulated, whereas post-implantationfactors become upregulated, methylation levels increase and one ofthe X chromosomes is inactivated in female cells (Nakamura et al.,2016; Xiang et al., 2019; Kalkan and Smith, 2014). Importantly, exitfrom the naïve pluripotent state happens concomitantly with themorphological transformation of the epiblast into an epithelial tissuethat lines the amniotic cavity, but whether these two events aremechanistically linked was unknown until recently. Using 3Dcultures of mouse and human ESCs, and mouse and humanembryo cultures, it has been shown that amniotic cavity formationis transcriptionally controlled by a pluripotent state transition(Shahbazi et al., 2017). Naïve pluripotent cells polarise in responseto the underlying ECM, but apical vesicle exocytosis is compromised.Consequently, failure to dismantle the naïve state leads to impairedamniotic cavitation and epiblast epithelialisation both in mouse andhuman embryos (Shahbazi et al., 2017). In the mouse epiblast, thetranscription factors Oct4 and Otx2 induce the expression ofpodocalyxin, which participates in lumen opening by promotingelectrostatic membrane repulsion of apposing membranes duringvesicle exocytosis. This transcriptional regulation may not beconserved in human embryos, as OTX2 is weakly expressed in thehuman epiblast (Xiang et al., 2019), andOCT4 has different functionsin mouse and human blastocysts (Fogarty et al., 2017). Therefore, thetranscription factors that control amniotic cavity formation in humanembryos remain to be determined. Notwithstanding the specificmechanism, this is a very clear example of how a cell identity changeis necessary for a change in tissue shape.The fate-shape relationship is bidirectional, as changes in cell

shape and mechanics also control pluripotent cell identity.

Experiments in human ESCs revealed that epithelialisation leadsto decreased pluripotent gene expression via integrin β1 activation(Hamidi et al., 2020), and therefore epiblast epithelialisation couldfacilitate pluripotency exit in vivo. In mouse ESCs, a decrease inmembrane tension is necessary for naïve pluripotency exit (Bergertet al., 2019), as it allows the activation of FGF signalling, a trigger ofnaïve exit, via endocytosis of FGF receptors (De Belly et al., 2019).In human ESCs FGF signalling inhibition is implied as a keyrequirement for naïve human ESCs, and its activation is associatedwith naïve pluripotency exit (Di Stefano et al., 2018). Thus, it istempting to speculate that loss of the naïve state in human embryoscould also be regulated by changes in membrane tension leading toFGF receptor endocytosis (Fig. 3). Future studies are needed todetermine whether similar changes in membrane tension andsignalling are observed beyond 2D cultures, using systems thatrecapitulate both epiblast gene expression and shape. Cell-cell andcell-matrix interactions, as well as the mechanical properties of theECM, are major regulators of pluripotent stem cell identity in vitro(Ranga et al., 2014; Przybyla et al., 2016; Pieters and Van Roy, 2014;Hamidi et al., 2020). Upon naïve pluripotency exit, cells becomeresponsive to mechanical stimuli. In particular, cell stretching leads toan increase in the intracellular levels of calcium, which activate stressand metabolic pathways (Verstreken et al., 2019).

Altogether, these studies provide a glimpse of the complexinterplay between transcriptional responses, mechanics, geometryand signalling in pluripotent cells. However, functional studies in thehuman embryo are needed to determine whether these mechanismsare conserved from ESCs to embryos and from mice to humans.

Amnion differentiationA key event in the development of implanting human embryos is thesplit of epiblast cells into the pluripotent epiblast disc and thedifferentiated extra-embryonic amniotic epithelium. Our knowledgeof the mechanisms that govern this event is very rudimentary, as inmost animal models amnion formation takes place at gastrulation(Dobreva et al., 2010). Much of what we know is derived fromstudies in monkey embryos (Hill, 1932). Upon implantation,epiblast cells in contact with the cytotrophoblast adopt asquamous epithelial morphology and downregulate pluripotencyfactors such as NANOG and SOX2, whereas epiblast cells incontact with the hypoblast form a columnar epithelium and remainpluripotent (Luckett, 1975; Sasaki et al., 2016). This fate split couldbe regulated by a gradient of BMP and/or WNT activity, as themonkey cytotrophoblast and amnion are a source ofWNT and BMPligands, whereas the hypoblast secretes WNT and BMP inhibitors(Sasaki et al., 2016). In agreement with this notion, stimulation ofhuman ESCs with BMP leads to the formation of amnion-like cells(Guo et al., 2020; Minn et al., 2020), BMP signalling is required foramnion specification in mouse embryos (Dobreva et al., 2018) andculture of human ESCs in a soft 3D matrix generates amnion-likesquamous spheroids in a BMP-dependent manner (Shao et al.,2016). Mechanical cues may cooperate with BMP signalling, asplating human ESCs on a hard surface prevents amniogenesis (Shaoet al., 2016). Amnion formation is also regulated by cell density;high cell densities preserve pluripotency and inhibit amniogenesis,whereas low cell densities promote amnion specification (Shaoet al., 2017). However, the exact mechanism of amnionspecification awaits further investigation.

Gastrulation: from pluripotency loss to lineage specificationThe day 14 rule limits human embryo research to the first 14 days ofdevelopment (Pera, 2017) and, thus, to the period prior to the

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formation of the primitive streak (see Glossary, Box 1).Consequently, gastrulation cannot be studied in human embryos.Given that it is ‘not birth, marriage or death, but gastrulation whichis truly the most important time in your life’ (Wolpert and Vicente,2015), it is not surprising that multiple groups are putting a greateffort into recreating human embryogenesis using stem cells(Shahbazi et al., 2019; Simunovic and Brivanlou, 2017). Duringgastrulation, cells undergo concomitant changes in shape and fate.At the site where the primitive streak forms, epiblast cells undergoepithelial-to-mesenchymal transition (EMT) (see Glossary, Box 1),lose their pluripotent features and generate mesoderm (see Glossary,Box 1) and endoderm (see Glossary, Box 1) (Tam and Behringer,1997). Epiblast progenitors that do not ingress through the streakbecome ectoderm (see Glossary, Box 1). The onset of gastrulationis regulated by the signalling crosstalk between embryonic andextra-embryonic cells, mediated by the concerted action of BMP,WNT and activin A/Nodal signals and their inhibitors (Tam andLoebel, 2007). These signalling interactions may be furthermodulated by mechanical cues and the physical structure of thetissue. Modelling the human embryo using stem cells disentanglesthis complexity by modulating single parameters at a time (Siggiaand Warmflash, 2018).

Self-organisation of human ESCsIn human ESC cultures, cell density and colony shape and size canbe precisely controlled using micropattern technology (Théry andPiel, 2009; Bauwens et al., 2008; Nazareth et al., 2013). Theseinitial studies led to the conclusion that colony size and compositionaffect human ESC identity in the absence of exogenous cues(Peerani et al., 2007).Subsequent experiments applied micropattern technology to the

study of tissue patterning. When human ESCs are cultured inmicropatterns of 0.5 mm, they reach a cell density equivalent to that

observed in the pre-gastrulating human epiblast (Etoc et al., 2016).Addition of BMP4 triggers pluripotency exit and lineagecommitment. Despite the homogenous presentation of themorphogen, germ layers become radially organised, with TE andamnion on the outside, followed by endoderm, mesoderm andectoderm towards the centre of the colony (Warmflash et al., 2014;Minn et al., 2020). Mechanistic studies revealed that patterning wasa consequence of the diffusion of the BMP4 inhibitor noggin(NOG) and the epithelial architecture of the colony (Etoc et al.,2016). As a result of the epithelial nature of human ESCs, BMPreceptors become basally localised and therefore inaccessible to theapically presented BMP4. However, cells in the border of the colonyhave a less pronounced polarised phenotype, which allows ligand-receptor interactions and, hence, differentiation of the outermostcells. Negative feedback by NOG, which is directly induced byBMP4, is also required to explain the fate patterning (Etoc et al.,2016). Interestingly, a recent report has shown that BMP receptorsare basolaterally localised in the early post-implantation mouseepiblast, and this is required for the formation of a robust BMPsignalling gradient (Zhang et al., 2019), indicating tissue geometrycontrols pluripotent cell differentiation both in mice and humans(Fig. 4).

An alternative mechanistic explanation for the emergence ofpatterning upon BMP4 stimulation has been proposed by Tewaryet al. (2017). A reaction-diffusionmechanism is initially responsiblefor the generation of a self-organised BMP4 gradient. Thesubsequent interpretation of that gradient and the patterned fateacquisition is consistent with the positional information model. Insupport of this idea, decreasing the concentration of BMP4 issufficient to preserve fate patterning in micropatterns of smaller size(0.2-0.3 mm). This represents a potential mechanism to adapt tochanges in embryo size, and hence maintain developmentalrobustness (Tewary et al., 2017). However, a recent report has

Cell fate:gene expression changes

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RNA

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Increased FGF activity?

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Exocytosis

Lumenogenesis

Post-implantation factors

Endocytosis

Membrane tension

Transcription factors

Blastocyst Blastocyst

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Exocytosis and luminal proteins

Naive genes

Fig. 3. Proposed cell fate-tissue shape crosstalk during human implantation development. Upon implantation, epiblast cells exit from the naïvepluripotent state and initiate the expression of post-implantation factors. Studies in mouse ESCs have shown that this is controlled by a decrease in membranetension, which promotes endocytosis and, as a consequence, increased fibroblast growth factor (FGF) signalling activity, which is required for naïvepluripotency exit. Therefore, membrane tension affects the pluripotent state of a cell. Whether this pathway is active in human embryos remains to beexplored. Upon naïve pluripotency exit, genes involved in exocytosis become expressed and initiate the process of lumenogenesis to form the amnioticcavity. Exocytic vesicles provide apical membrane and luminal proteins, which are necessary to build a lumen de novo. The transcription factors involvedin this process remain to be identified. Question mark denotes a molecular event that has not beem validated in human embryos.

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proposed that BMP4 does not act as a classical morphogen. In verysmall colonies (one to eight cells), the response to BMP4 is binary:cells adopt either a pluripotent or a TE fate (Nemashkalo et al.,2017). The appearance of mesendodermal fates in a primitivestreak-like region additionally requires WNT and Nodal activation(Siggia and Warmflash, 2018).Self-organised patterns also emerge upon stimulation with WNT

(Martyn et al., 2018), which leads to the appearance of an outermesendoderm ring while inner cells remain pluripotent. Formationof this fate pattern requires E-cadherin cell-cell adhesions, which aremore prominent in the colony centre, and dampen WNT activity bysequestering β-catenin. In addition, the WNT inhibitor DKK1 isexpressed in response to WNT and controls the size of themesendoderm ring (Martyn et al., 2019). In agreement with thesemicropattern studies, a group of cells in the primitive streak ofmouse and rabbit embryos expresses both activators and inhibitorsof the BMP and WNT signalling pathways (Peng et al., 2016;Idkowiak et al., 2004), indicating there are epiblast-intrinsicmechanisms that regulate the position of the primitive streak.These examples demonstrate how simplified 2D models of the

embryo can help us dissect the chemical control of tissue patterning;but mechanical cues also play a role in patterning. For example,mechanical strains during gastrulation lead to mesodermspecification via induction of the transcription factor brachyury,and this mechanism may be conserved from flies to humans (Brunetet al., 2013). Although the 2D self-organisation of human ESCsdescribed so far does not recapitulate the mechanical features of thedeveloping embryo, recent technological advances provide anopportunity to explore the contribution of mechanics (Vianello andLutolf, 2019). The use of hydrogels led to the observation thatmatrix stiffness, degradability and composition influenceproliferation, tissue structure and differentiation in the neurallineage (Ranga et al., 2016; Sun et al., 2014). Similarly, mesodermand endoderm differentiation are enhanced in soft substrates butinhibited in stiffer ones (Przybyla et al., 2016; Chen et al., 2020).Combining micropattern technology with embryo-like compliant

hydrogels revealed that gastrulation-like domains emerge in areas ofhigh tension due to the release of β-catenin from cell-cell adhesionsites and its nuclear translocation, both in mouse and human ESCs(Muncie et al., 2020; Blin et al., 2018). To dissect the role ofmechanics during development, future experimental strategies needto: be based on the use of robust, reproducible and relevant stem cellmodels of the embryo (Huch et al., 2017; Van Den Brink et al.,2020); allow mapping of intrinsic stresses and application ofextrinsic forces in a precise manner (Vianello and Lutolf, 2019); anduse appropriate readouts to assess changes in patterning andmorphogenesis.

Embryonic-extra-embryonic crosstalkWe can add an extra layer of complexity to self-organising modelsof human ESCs by investigating how extra-embryonic cellsmodulate human embryonic stem cell fate and shape atgastrulation. This is particularly important given the differentorganisation of extra-embryonic tissues in mouse and humanembryos. Microfluidics has been recently used to model theepiblast-amnion fate split (Zheng et al., 2019). This led to thecharacterisation of the amnion as a crucial signalling centre.Amniotic epithelial-like cells express high levels of BMP4 andinduce gastrulation-like events in a WNT-dependent manner inpluripotent human ESCs and specification of primordial germ cell-like cells (see Glossary, Box 1) (Zheng et al., 2019). Therefore,whereas in mouse embryos the extra-embryonic ectoderm is the keydriver of gastrulation (Tam and Behringer, 1997), in humanembryos this role may be played by the amnion. Conversely, thefunction of the hypoblast as an inhibitor of BMP andWNT signallingseems to be conserved in mouse, monkey and humans (Xiang et al.,2019; Stower and Srinivas, 2014; Sasaki et al., 2016). Hypoblast cellssecrete BMP and WNT inhibitors in a polarised manner, andtherefore the combined action of TE/amnion and hypoblast leads tothe generation of a gradient of BMP and WNT activity across theepiblast. Future studies are needed to dissect the specificcontributions of the cytotrophoblast, hypoblast and amnion in

Cell fate:gene expression changes

Polarized mesoderm marker expression

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Morphogenesis:tissue shape changes

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EMT transcription factors

Decreased cell-cell adhesion

Brachyury

Unpolarised BMP receptors

Increased BMP and WNT signalling

Mesendoderm specification

Epiblast disc

Gastrula

Epiblast disc

Fig. 4. Proposed cell fate-tissue shape crosstalk during human post-implantation development. BMP and WNT signals secreted by the amnion and thetrophectoderm lead to expression of mesoderm markers, such as the transcription factor brachyury, in the posterior epiblast. Brachyury induces expression ofepithelial-to-mesenchymal transition (EMT) transcription factors, which cause a decrease in the levels of E-cadherin. As a consequence, cell-cell adhesionsweaken and cells lose their epithelial morphology and their polarised organisation, such as the polarised localisation of BMP receptors. The unpolarised BMPreceptors are able to interact with BMP ligands, increasing BMP signalling activity and inducing mesendoderm specification.

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patterning the human epiblast. This could be achieved by combiningstudies on in vitro cultured human embryos (Xiang et al., 2019) within vivo and in vitro cultured monkey embryos (Nakamura et al., 2016;Niu et al., 2019; Ma et al., 2019) and the development of new stemcell models of the human embryo comprising both embryonic andextra-embryonic stem cells, as previously used in the mouse(Harrison et al., 2017; Sozen et al., 2018; Rivron et al., 2018).

When things go wrong: aneuploidy and pregnancy lossUnderstanding the basic mechanisms of human development isfundamental to tackling an important clinical problem: theremarkable inefficiency of human reproduction. It is estimatedthat only 30-40% of conceptions progress to live birth (Larsen et al.,2013). Development may fail at any time between fertilisation andterm, but the early stages are especially crucial. Epidemiologicalstudies estimate that 40-50% of embryos are lost before implantation(Wilcox et al., 2020). Once the embryo implants, the ongoingpregnancy can be detected based on the increased levels of humanchorionic gonadotropin (hCG) (Wilcox et al., 1999). Based on hCGserum levels, 20-30% of implanted embryos fail to progress beyondweek 6 (Wilcox et al., 1988). Owing to the lack of ultrasoundverification, these cases are termed biochemical losses (Larsen et al.,2013;Macklon et al., 2002). The reasons behind this high rate of earlypregnancy loss remain poorly understood. Multiple potentialmechanisms have been involved, including both alterations inembryo development and maternal causes, but further research isneeded to understand their contribution.The observation that 50-75% of spontaneous abortions present

karyotypic abnormalities and morphological malformations(Philipp et al., 2003; Boué et al., 1975) has resulted in a focus onthe genetic causes of pregnancy loss. While the incidence of meioticaneuploidy in most species is remarkably low, 10-30% of fertilisedhuman eggs are aneuploid due to female meiotic errors (Hassoldand Hunt, 2001; Nagaoka et al., 2012). These chromosomesegregation errors are more prevalent in young women and inwomen of advanced age, resulting from whole-chromosomenondisjunction and premature sister chromatid separation,respectively (Gruhn et al., 2019). Why, when and how embryosharbouring specific aneuploidies fail during embryogenesis remainsunknown. All single chromosome aneuploidies can potentially formblastocysts, but they do so at a lower rate and with a higher incidenceof morphological abnormalities. Aneuploid embryos are morelikely to arrest during EGA, to be developmentally delayed and topresent a low-quality TE and/or ICM (i.e. few cells and cellularfragmentation indicative of cell death) (Minasi et al., 2016; Fragouliet al., 2013). Moreover, despite forming morphologically normalblastocysts, aneuploid embryos display transcriptional andepigenetic alternations that may compromise their subsequentdevelopment (McCallie et al., 2016; Licciardi et al., 2018; Starostiket al., 2020). Autosomal monosomies typically lead to biochemicallosses, whereas autosomal trisomies are associated with silentmiscarriage (presence of extra-embryonic membranes but lack ofembryonic structures) and first-trimester miscarriage (Ljunger et al.,2005; Martinez et al., 2010; Ferro et al., 2003; Stephenson et al.,2002). In addition, alterations in the number of chromosomes can bea consequence of mitotic alterations during the first three cleavagedivisions, which lead to the formation of mosaic human embryos(Popovic et al., 2019; Starostik et al., 2020; Mantikou et al., 2012).Given the inaccessibility of human embryos at these stages, severalfundamental questions remain to be addressed. How do specificalterations in the number of chromosomes impact onmorphogenesisor cell fate specification? Is the response to aneuploidy different in

embryonic and extra-embryonic cells? What is the fate of aneuploidcells in mosaic embryos? Healthy babies can potentially be bornfrom mosaic aneuploid human embryos (Greco et al., 2015),suggesting aneuploid cells may be selectively eliminated duringdevelopment. Could this selective elimination be a consequence ofthe impaired response of aneuploid cells to physical and/orbiochemical cues? And given that 25-50% of spontaneousmiscarriages do not show chromosomal abnormalities, what is thereason behind their failed development? Could alterations in the cellfate-tissue shape crosstalk be responsible for some of these earlypregnancy losses? Addressing these questions and understandingthe mechanisms that lead to pregnancy loss is a first step towards thefuture development of therapeutic interventions.

ConclusionsDespite remarkable advances in the field of human development, untilvery recently human embryo research was mainly descriptive and, asWilhelm Roux already realised by the end of the 19th century, tocomprehend the mechanisms of development, we must interfere withit. Today, we are ideally suited to tackle this challenge and decipherhow form and function emerge during human embryogenesis. Recentevidence indicates that the crosstalk between cell fate and tissue shapeplays an important role during key human developmental events,including compaction and polarisation in pre-implantation embryos,embryonic epithelialisation at the time of implantation and germlineage commitment at gastrulation. We are just beginning tounderstand how cell fate specification events modulate tissueorganisation and, vice versa, how changes in tissue shape modulatecell identity and tissue patterning, leading to the emergence of self-organisation. A number of crucial questions still remain to beaddressed. What are the mechanisms that remodel the implantinghuman embryo? How do physical and chemical cues coordinatemorphogenesis and patterning? What signals trigger cell fatespecification during gastrulation? And how are all these eventsaffected when chromosomal alterations are present? To address thesequestions, we now possess an adequate set of experimental tools:genome editing has already been successfully used to study genefunction in human embryos (Fogarty et al., 2017); novel imagingtechniques allow us to map cellular behaviours with unprecedentedresolution (McDole et al., 2018); new culture methods have extendedthe window of human development amenable to investigation(Shahbazi et al., 2016; Deglincerti et al., 2016; Xiang et al., 2019);and human ESC-embryo chimeras could be potentially used as a goldstandard to study cell potency (De Los Angeles et al., 2015;Alexandrova et al., 2016; Tachibana et al., 2012; Chen et al., 2015),although stringent criteria to assess lineage contribution and ethicalconsiderations need to be applied (Posfai et al., 2020). In addition,human ESCs can mimic different aspects of embryo development invitro (Shahbazi et al., 2019). These stem cell models of the embryo areallowing researchers to deconstruct the complexity of humandevelopment and to decipher the basic principles of self-organisation. Last and importantly, the overall increase in assistedreproductive technology cycles over the years, together with theimprovement in embryo selection procedures, leads to the generationof vast numbers of surplus human embryos (Kushnir et al., 2017),which represent an invaluable resource for research. Using this newset of tools, we can start to unravel the mysteries of our owndevelopment.

AcknowledgementsI am grateful to Eric Siggia, Meng Zhu, Bailey Weatherbee, Kirsty Mackinlay andMichele Petruzzelli for critical reading of the manuscript.

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Competing interestsThe authors declare no competing or financial interests.

FundingWork in M.N.S.’s group is funded by the Medical Research Council (MC_UP_1201/24) and the European Molecular Biology Organization. Deposited in PMC forimmediate release.

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