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Embryonic cells grow in environments that provide a plethora of physical cues, including mechanical forces that shape the development of the entire embryo. Despite their prevalence, the role of these forces in embryonic development and their integration with chemical signals have been mostly neglected, and scrutiny in modern molecular embryology tilted, instead, towards the dissection of molecular pathways involved in cell fate determination and patterning. It is now possible to investigate how mechanical signals induce downstream genetic regulatory networks to regulate key developmental processes in the embryo. Here, we review the insights into mechanical control of early vertebrate development, including the role of forces in tissue patterning and embryonic axis formation. We also highlight recent in vitro approaches using individual embryonic stem cells and self-organizing multicellular models of human embryos, which have been instrumental in expanding our understanding of how mechanics tune cell fate and cellular rearrangements during human embryonic development. Cells in the embryo are subject to autonomous and external mechanical forces that help steer embryonic tissue patterning. Technical developments, such as in vitro models of early embryos, allow probing of the roles of mechanical forces in animal and human embryonic development.

Chromosomal instability leading to aneuploidy is pervasive in early human embryos1-3 and is considered as a major cause of infertility and pregnancy wastage4,5. Here we provide several lines of evidence that blastocysts containing aneuploid cells are worthy of in vitro fertilization transfer. First, we show clinically that aneuploid embryos can lead to healthy births, suggesting the presence of an in vivo mechanism to eliminate aneuploidy. Second, early development and cell specification modelled in micropatterned human 'gastruloids' grown in confined geometry show that aneuploid cells are depleted from embryonic germ layers, but not from extraembryonic tissue, by apoptosis in a bone morphogenetic protein 4 (BMP4)-dependent manner. Third, a small percentage of euploid cells rescues embryonic tissue in mosaic gastruloids when mixed with aneuploid cells. Finally, single-cell RNA-sequencing analysis of early human embryos revealed a decline of aneuploidy beginning on day 3. Our findings challenge two current dogmas: that a single trophectoderm biopsy at blastocyst stage to perform prenatal genetic testing can accurately determine the chromosomal make-up of a human embryo, and that aneuploid embryos should be withheld from embryo transfer in association with in vitro fertilization.

As it fulfills an irresistible need to understand our own origins, research on human development occupies a unique niche in scientific and medical research. In this Comment, we explore the progress in our understanding of human development over the past 10 years. The focus is on basic research, clinical applications, and ethical considerations.

Harnessing the potential of human embryonic stem cells to mimic normal and aberrant development with standardized models is a pressing challenge. Here we use micropattern technology to recapitulate early human neurulation in large numbers of nearly identical structures called neuruloids. Dual-SMAD inhibition followed by bone morphogenic protein 4 stimulation induced self-organization of neuruloids harboring neural progenitors, neural crest, sensory placode and epidermis. Single-cell transcriptomics unveiled the precise identities and timing of fate specification. Investigation of the molecular mechanism of neuruloid self-organization revealed a pulse of pSMAD1 at the edge that induced epidermis, whose juxtaposition to central neural fates specifies neural crest and placodes, modulated by fibroblast growth factor and Wnt. Neuruloids provide a unique opportunity to study the developmental aspects of human diseases. Using isogenic Huntington’s disease human embryonic stem cells and deep neural network analysis, we show how specific phenotypic signatures arise in our model of early human development as a consequence of mutant huntingtin protein, outlining an approach for phenotypic drug screening. Micropatterning generates the four ectodermal cell types of human neurulation.

Breaking the anterior-posterior symmetry in mammals occurs at gastrulation. Much of the signalling network underlying this process has been elucidated in the mouse; however, there is no direct molecular evidence of events driving axis formation in humans. Here, we use human embryonic stem cells to generate an in vitro three-dimensional model of a human epiblast whose size, cell polarity and gene expression are similar to a day 10 human epiblast. A defined dose of BMP4 spontaneously breaks axial symmetry, and induces markers of the primitive streak and epithelial-to-mesenchymal transition. We show that WNT signalling and its inhibitor DKK1 play key roles in this process downstream of BMP4. Our work demonstrates that a model human epiblast can break axial symmetry despite the absence of asymmetry in the initial signal and of extra-embryonic tissues or maternal cues. Our three-dimensional model is an assay for the molecular events underlying human axial symmetry breaking.

In amniotes, the development of the primitive streak and its accompanying 'organizer' define the first stages of gastrulation. Although these structures have been characterized in detail in model organisms, the human primitive streak and organizer remain a mystery. When stimulated with BMP4, micropatterned colonies of human embryonic stem cells self-organize to generate early embryonic germ layers 1 . Here we show that, in the same type of colonies, Wnt signalling is sufficient to induce a primitive streak, and stimulation with Wnt and Activin is sufficient to induce an organizer, as characterized by embryo-like sharp boundary formation, markers of epithelial-to-mesenchymal transition and expression of the organizer-specific transcription factor GSC. Moreover, when grafted into chick embryos, human stem cell colonies treated with Wnt and Activin induce and contribute autonomously to a secondary axis while inducing a neural fate in the host. This fulfils the most stringent functional criteria for an organizer, and its discovery represents a milestone in human embryology.

Embryonic stem cells derived from the inner cell mass can differentiate to all embryonic lineages of any adult cell type and to a limited extent to extra-embryonic tissues. A study now allows the generation of pig and human stem cells with enhanced differentiation potential towards all embryonic and extra-embryonic fates, one step closer to the totipotent state of the fertilized egg.

Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions for the rapid differentiation of hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of 6 pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 days of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.

Embryos allocate cells to the three germ layers in a spatially ordered sequence. Human embryonic stem cells (hESCs) can generate the three germ layers in culture; however, differentiation is typically heterogeneous and spatially disordered. We show that geometric confinement is sufficient to trigger self-organized patterning in hESCs. In response to BMP4, colonies reproducibly differentiated to an outer trophectoderm-like ring, an inner ectodermal circle and a ring of mesendoderm expressing primitive-streak markers in between. Fates were defined relative to the boundary with a fixed length scale: small colonies corresponded to the outer layers of larger ones. Inhibitory signals limited the range of BMP4 signaling to the colony edge and induced a gradient of Activin-Nodal signaling that patterned mesendodermal fates. These results demonstrate that the intrinsic tendency of stem cells to make patterns can be harnessed by controlling colony geometries and provide a quantitative assay for studying paracrine signaling in early development.

Ref. 7 from Benvenisty and colleagues was inadvertently omitted; this has now been cited in the text and added to the reference list, and subsequent references have been renumbered. The Letter has been corrected online.

Cancer cells have the ability to divide indefinitely and spread to different parts of the body during metastasis. Embryonic stem cells can self-renew and, through differentiation to somatic cells, provide the building blocks of the human body. Embryonic stem cells offer tremendous opportunities for regenerative medicine and serve as an excellent model system to study early human development. Many of the molecular mechanism underlying tumorigenesis in cancer and self-renewal in stem cells have been elucidated in the past decade. Here we present a systematic analysis of seven major signaling pathways implicated in both cancer and stem cells. We present on overview of the JAK/STAT, Notch, MAPK/ERK, PI3K/AKT, NF-kB, Wnt and TGF-beta pathways and analyze their activation status in the context of cancer and stem cells. We focus on their role in stem cell self-renewal and development and identify key molecules, whose aberrant expression has been associated with malignant phenotypes. We conclude by presenting a map of the signaling networks involved in cancer and embryonic stem cells.

Human and mouse embryonic stem cells (HESCs and MESCs, respectively) self-renew indefinitely while maintaining the ability to generate all three germ-layer derivatives. Despite the importance of ESCs in developmental biology and their potential impact on tissue replacement therapy, the molecular mechanism underlying ESC self-renewal is poorly understood. Here we show that activation of the canonical Wnt pathway is sufficient to maintain self-renewal of both HESCs and MESCs. Although Stat-3 signaling is involved in MESC self-renewal, stimulation of this pathway does not support self-renewal of HESCs. Instead we find that Wnt pathway activation by 6-bromoindirubin-3'-oxime (BIO), a specific pharmacological inhibitor of glycogen synthase kinase-3 (GSK-3), maintains the undifferentiated phenotype in both types of ESCs and sustains expression of the pluripotent state-specific transcription factors Oct-3/4, Rex-1 and Nanog. Wnt signaling is endogenously activated in undifferentiated MESCs and is downregulated upon differentiation. In addition, BIO-mediated Wnt activation is functionally reversible, as withdrawal of the compound leads to normal multidifferentiation programs in both HESCs and MESCs. These results suggest that the use of GSK-3-specific inhibitors such as BIO may have practical applications in regenerative medicine.

Neural induction represents the earliest step in the determination of ectodermal cell fates. In vertebrates, bone morphogenetic proteins (BMPs) act as signals of epidermal induction. The inhibition of the BMP signalling pathway in the ectoderm is the hallmark of neural-fate acquisition, and forms the basis of the default model of neural induction. BMP inhibition seems to take place through distinct mechanisms in different vertebrate species, including transcriptional regulation of BMP gene expression and clearance of BMP ligands by secreted inhibitors. Here, we discuss the role of fibroblast growth factors and Wnt proteins in neural induction and in the regulation of BMP signalling in the ectoderm of Xenopus laevis and chick embryos. In addition, we discuss evidence from mouse embryonic stem cells that supports the default model of neural induction and the role of BMP signalling in ectodermal fate determination.

Bone morphogenetic proteins (BMPs), including the fly homologue Decapentaplegic (DPP), are important regulators of early vertebrate and invertebrate dorsal–ventral development1,2,3,4,5,6. An evolutionarily conserved BMP regulatory mechanism operates from fly to fish, frog and mouse to control the dorsal–ventral axis determination. Several secreted factors, including the BMP antagonist chordin/Short gastrulation (SOG)7,8,9,10,11,12, modulate the activity of BMPs. In Drosophila, Twisted gastrulation (TSG) is also involved in dorsal–ventral patterning13,14,15, yet the mechanism of its function is unclear. Here we report the characterization of the vertebrate Tsg homologues. We show that Tsg can block BMP function in Xenopus embryonic explants and inhibits several ventral markers in whole-frog embryos. Tsg binds directly to BMPs and forms a ternary complex with chordin and BMPs. Coexpression of Tsg with chordin leads to a more efficient inhibition of the BMP activity in ectodermal explants. Unlike other known BMP antagonists, however, Tsg also reduces several anterior markers at late developmental stages. Our data suggest that Tsg can function as a BMP inhibitor in Xenopus; furthermore, Tsg may have additional functions during frog embryogenesis.

The stem-cell meeting held at the Institut Pasteur in December 2004 proved that the nascent field of stem-cell biology has rapidly become one of the most exciting and active fields in current research. Leading scientists from a range of disciplines relevant to stem-cell biology covered a gamut of current topics, including the properties of human and mouse embryonic and adult stem cells, attempts to manipulate stem cells, and the first clinical attempts at cell-based therapies. Here we describe some of the high points.

Nat. Neurosci. 5, 931 (2002). The authors wish to correct the sentence in paragraph 4 on page 931, which should read “Moreover, chapter 5 of this book is nearly identical to chapter 9 of the MIT Press book, including identical figures.”

A report on the Eighth Biannual Xenopus Conference, Estes Park, Colorado, August 16-20, 2000.