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Embryonic tissue boundaries are critical to not only cement newly patterned structures during development, but also to serve as organizing centers for subsequent rounds of morphogenesis. ​Although this latter role is especially difficult to study in vivo, synthetic embryology offers a new vantage point and fresh opportunities. In this review, we cover recent progress towards understanding and controlling in vitro boundaries and how they impact synthetic model systems. A key point this survey highlights is that the outcome of self-organization is strongly dependent on the boundary imposed, and new insight into the complex functions of embryonic boundaries will be necessary to create better self-organizing tissues for basic science, drug development, and regenerative medicine.

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.

The combination of affordability, large size, and ease of access at almost every stage of development renders the chick an excellent model organism for studying vertebrate development. Not only is it a great system in and of itself, but these qualities make it a great host for interspecies chimera experiments. In this chapter we highlight some notable examples of mammalian-chick chimeras, and show how one can for instance use the chick to push mammalian stem cell experiments further to learn about the behavior and capabilities of these cells in vivo. In particular, here we present the methodology necessary for transplantation of human embryonic stem cell (hESC)-derived "gastruloids" stimulated to generate a human organizer into the chick embryo. In these human-chick chimeras, the human organizer cells self-organize to contribute directly to notochord-like tissue and indirectly induce host chick cells to generate neural tissue.

Although fate maps of early gastrula embryos exist for nearly all model organisms, a fate map of the gastrulating human embryo remains elusive. Here we use human gastruloids to piece together part of a rudimentary fate map of the human primitive streak (PS). This is possible because stimulation with differing levels of BMP, WNT, and NODAL leads to self-organization of gastruloids into large and homogenous different subpopulations of endoderm and mesoderm, and comparative parallel analysis of these gastruloids, together with the fate map of the mouse embryo, allows the organization of these subpopulations along an anterior-posterior axis. We also developed a novel cell tracking technique that allowed the detection of robust fate-dependent cell migrations in our gastruloids comparable to those found in the mouse embryo. Taken together, our gastruloid derived fate map and recording of cell migrations provides a first coarse view of the embryonic human PS.

Although fate maps of early embryos exist for nearly all model organisms, a fate map of the gastrulating human embryo remains elusive. Here we use human gastruloids to piece together a rudimentary fate map for the human primitive streak (PS). This is possible because differing levels of BMP, WNT, and NODAL leads to self-organization of gastruloids into homogenous subpopulations of endoderm and mesoderm, and comparative analysis of these gastruloids, together with the fate map of the mouse embryo, allows the organization of these subpopulations along an anterior-posterior axis. We also developed a novel cell tracking technique that detected robust fate-dependent cell migrations in our gastruloids comparable to those found in the mouse embryo. Taken together, our fate map and recording of cell migrations provides a first coarse view of what the human PS may resemble in vivo.

The combination of affordability, large size, and ease of access at almost every stage of development renders the chick an excellent model organism for studying vertebrate development. Not only is it a great system in and of itself, but these qualities make it a great host for interspecies chimera experiments. In this chapter we highlight some notable examples of mammalian-chick chimeras, and show how one can for instance use the chick to push mammalian stem cell experiments further to learn about the behavior and capabilities of these cells in vivo. In particular, here we present the methodology necessary for transplantation of human embryonic stem cell (hESC)-derived "gastruloids" stimulated to generate a human organizer into the chick embryo. In these human-chick chimeras, the human organizer cells self-organize to contribute directly to notochord-like tissue and indirectly induce host chick cells to generate neural tissue.

Long range signalling by morphogens and their inhibitors define embryonic patterning yet quantitative data and models are rare, especially in humans. Here we use a human embryonic stem cell “gastruloid” system to model formation of the primitive streak (PS) by WNT. In the pluripotent state E-CADHERIN (E-CAD) transduces boundary forces to focus WNT signalling to colony border. Following application of WNT ligand, E-CAD mediates a wave of epithelial-to-mesenchymal (EMT) conversion analogous to PS extension in an embryo. By knocking out the secreted WNT inhibitors active in our system, we show that DKK1 alone controls the extent and duration of patterning. The NODAL inhibitor CERBERUS1 acts downstream of WNT to refine the endoderm versus mesoderm fate choice. Our EMT wave is a generic property of a bistable system with diffusion and a single quantitative model describes both the wave and our knockout data.

Self-organization of discrete fates in human gastruloids is mediated by a hierarchy of signaling pathways. How these pathways are integrated in time, and whether cells maintain a memory of their signaling history remains obscure. Here, we dissect the temporal integration of two key pathways, WNT and ACTIVIN, which along with BMP control gastrulation. CRISPR/Cas9-engineered live reporters of SMAD1, 2 and 4 demonstrate that in contrast to the stable signaling by SMAD1, signaling and transcriptional response by SMAD2 is transient, and while necessary for pluripotency, it is insufficient for differentiation. Pre-exposure to WNT, however, endows cells with the competence to respond to graded levels of ACTIVIN, which induces differentiation without changing SMAD2 dynamics. This cellular memory of WNT signaling is necessary for ACTIVIN morphogen activity. A re-evaluation of the evidence gathered over decades in model systems, re-enforces our conclusions and points to an evolutionarily conserved mechanism., eLife digest Embryonic stem cells can renew themselves to generate more stem cells, or specialize to become any type of cell found in an adult. They therefore hold great potential for studying how we develop from a single cell into a complex organism made of many different cell types. In a key stage of development, individual cells form into organized tissues. The earliest phase of tissue organization involves the formation of three ‘germ layers’. Human embryonic stem cells allow us to recreate this early stage of embryo development in the lab. When grown in confined spaces, the cells organize into clusters that can then develop germ layers. Previous work using these clusters showed that a network of signaling proteins – including one called WNT – trigger human embryonic stem cells to form the initial clusters. Then, another signaling protein called ACTIVIN tells the cells to specialize to form the two inner germ layers. But in experiments that apply only the ACTIVIN signal, the cells instead keep dividing to make more stem cells. ACTIVIN can trigger the activity of a protein called SMAD. To visualize how cells respond to ACTIVIN in real time, Yoney et al. used a gene editing technique called CRISPR to add fluorescent tags to SMAD in human embryonic stem cells. The results show that the ACTIVIN response triggers a peak in the amount of SMAD in the cell’s nucleus that then decreases over several hours. This briefly activated several genes that are known to help to form germ layers. However, this gene activity was not maintained for long enough to cause the stem cells to specialize and organize into layers. Yoney et al. then repeated the experiments on cells that had previously been exposed to WNT signaling proteins. The germ layer gene activity was maintained in this case, leading to the cells specializing and forming the inner two germ layers. This suggests that the cells somehow remembered the WNT signal, and this memory changed how they responded to ACTIVIN. The next step is to understand how cells store the memory of the WNT signal. As well as aiding our understanding of development, it could also help us to understand situations where signaling goes wrong, such as cancer. The technique used here to follow signals in real time could also be used to study other biological signaling processes.

Breaking the anterior-posterior (AP) symmetry in mammals takes place at gastrulation. Much of the signaling 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 3D model of a human epiblast whose size, cell polarity, and gene expression are similar to a 10-day human epiblast. A defined dose of bone mor-phogenetic protein 4 (BMP4) spontaneously breaks axial symmetry, and induces markers of the primitive streak and epithelial to mesenchymal transition. By gene knockouts and live-cell imaging we show that, downstream of BMP4, WNT3 and its inhibitor DKK1 play key roles in this process. Our work demonstrates that a model human epiblast can break axial symmetry despite no asymmetry in the initial signal and in the absence of extraembryonic tissues or maternal cues. Our 3D model opens routes to capturing molecular events underlying axial symmetry breaking phenomena, which have largely been unexplored in model human systems.

In amniotes, the development of the primitive streak (PS) and its accompanying “organizer” define the first stages of gastrulation. Despite detailed characterization in model organisms, the analogous human structures remain a mystery. We have previously shown that when stimulated with BMP4, micropatterned colonies of human embryonic stem cells (hESCs) self-organize to generate early embryonic germ layers1. Here we show that in the same type of colonies WNT signalling is sufficient to induce a PS, and WNT with ACTIVIN is sufficient to induce an organizer, as characterized by embryo-like sharp boundary formation, epithelial-to-mesenchymal transition (EMT) markers, and expression of the organizer specific transcription factor GSC. Moreover, when grafted into chick embryos, WNT and ACTIVIN treated human cells induce and contribute autonomously to a secondary axis while inducing neural fate in the host. This fulfills the most stringent functional criteria for an organizer, and its discovery represents a major milestone in human embryology.

Fate allocation in the gastrulating embryo is spatially organized as cells differentiate into specialized cell types depending on their positions with respect to the body axes. There is a need for in vitro protocols that allow the study of spatial organization associated with this developmental transition. Although embryoid bodies and organoids can exhibit some spatial organization of differentiated cells, methods that generate embryoid bodies or organoids do not yield consistent and fully reproducible results. Here, we describe a micropatterning approach in which human embryonic stem cells are confined to disk-shaped, submillimeter colonies. After 42 h of BMP4 stimulation, cells form self-organized differentiation patterns in concentric radial domains, which express specific markers associated with the embryonic germ layers, reminiscent of gastrulating embryos. Our protocol takes 3 d; it uses commercial microfabricated slides (from CYTOO), human laminin-521 (LN-521) as extracellular matrix coating, and either conditioned or chemically defined medium (mTeSR). Differentiation patterns within individual colonies can be determined by immunofluorescence and analyzed with cellular resolution. Both the size of the micropattern and the type of medium affect the patterning outcome. The protocol is appropriate for personnel with basic stem cell culture training. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation.

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.

In molecular systematics, evolutionary trees are reconstructed from sequences at the tips under simple models of site substitution. A central question is how much sequence data is required to reconstruct a tree accurately? The answer depends on the lengths of the branches (edges) of the tree, with very short and very long edges requiring long sequences for accurate tree inference, particularly when these branch lengths are arranged in certain ways. For four-taxon trees, the sequence length question was settled for the case of a rapid speciation event in the distant past. Here, we generalize this result and show that the same sequence length requirement holds even when the speciation event is recent, provided that at least one of the four taxa is distantly related to the others. However, this equivalence disappears if a molecular clock applies, since the length of the long outgroup edge becomes largely irrelevant in the estimation of the tree topology for a recent (but not a deep) divergence. We also show how our results can be extended to models in which substitution rates vary across sites, and to settings where more than four taxa are involved., Comment: 20 pages, 2 figures

We present optimal linear time algorithms for computing the Shapley values and 'heightened evolutionary distinctiveness' (HED) scores for the set of taxa in a phylogenetic tree. We demonstrate the efficiency of these new algorithms by applying them to a set of 10,000 reasonable 5139-species mammal trees. This is the first time these indices have been computed on such a large taxon and we contrast our finding with an ad-hoc index for mammals, fair proportion (FP), used by the Zoological Society of London's EDGE programme. Our empirical results follow expectations. In particular, the Shapley values are very strongly correlated with the FP scores, but provide a higher weight to the few monotremes that comprise the sister to all other mammals. We also find that the HED score, which measures a species' unique contribution to future subsets as function of the probability that close relatives will go extinct, is very sensitive to the estimated probabilities. When they are low, HED scores are less than FP scores, and approach the simple measure of a species' age. Deviations (like the Solendon genus of the West Indies) occur when sister species are both at high risk of extinction and their clade roots deep in the tree. Conversely, when endangered species have higher probabilities of being lost, HED scores can be greater than FP scores and species like the African elephant Loxondonta africana, the two solendons and the thumbless bat Furipterus horrens can move up the rankings. We suggest that conservation attention be applied to such species that carry genetic responsibility for imperiled close relatives. We also briefly discuss extensions of Shapley values and HED scores that are possible with the algorithms presented here.

In the face of inevitable future losses to biodiversity, ranking species by conservation priority seems more than prudent. Setting conservation priorities within species (i.e., at the population level) may be critical as species ranges become fragmented and connectivity declines. However, existing approaches to prioritization (e.g., scoring organisms by their expected genetic contribution) are based on phylogenetic trees, which may be poor representations of differentiation below the species level. In this paper we extend evolutionary isolation indices used in conservation planning from phylogenetic trees to phylogenetic networks. Such networks better represent population differentiation, and our extension allows populations to be ranked in order of their expected contribution to the set. We illustrate the approach using data from two imperiled species: the spotted owl Strix occidentalis in North America and the mountain pygmy-possum Burramys parvus in Australia. Using previously published mitochondrial and microsatellite data, we construct phylogenetic networks and score each population by its relative genetic distinctiveness. In both cases, our phylogenetic networks capture the geographic structure of each species: geographically peripheral populations harbor less-redundant genetic information, increasing their conservation rankings. We note that our approach can be used with all conservation-relevant distances (e.g., those based on whole-genome, ecological, or adaptive variation) and suggest it be added to the assortment of tools available to wildlife managers for allocating effort among threatened populations.