At the onset of mammalian gastrulation, secreted signalling molecules belonging to the Bmp, Wnt, Nodal and Fgf signalling pathways induce and pattern the primitive streak, marking the start for the cellular rearrangements that generate the body plan. Our current understanding of how signalling specifies and organises the germ layers in three dimensions, was mainly derived from genetic experimentation using mouse embryos performed over many decades. However, the exact spatiotemporal sequence of events is still poorly understood, both because of a lack of tractable models that allow for real time visualisation of signalling and differentiation and because of the molecular and cellular complexity of these early developmental events. In recent years, a new wave of in vitro embryo models has begun to shed light on the dynamics of signalling during primitive streak formation. Here we discuss the similarities and differences between a widely adopted mouse embryo model, termed gastruloids, and real embryos from a signalling perspective. We focus on the gene regulatory networks that underlie signalling pathway interactions and outline some of the challenges ahead. Finally, we provide a perspective on how embryo models may be used to advance our understanding of signalling dynamics through computational modelling.

Microtubule-actin cross-linking factor 1 (MACF1), also known as actin cross-linking family protein 7 (ACF7), is a giant cytolinker protein with multiple conserved domains that can orchestrate cytoskeletal networks of actin and microtubules. MACF1 is involved in various biological processes, including cell polarity, cell-cell connection, cell proliferation, migration, vesicle transport, signal transduction, and neuronal development. In this review, we updated the physiological and pathological roles of MACF1, highlighting the components and signaling pathways involved. Novel evidence showed that MACF1 is involved in diverse human diseases, including multiple neuronal diseases, congenital myasthenic syndrome, premature ovarian insufficiency, spectraplakinopathy, osteoporosis, proliferative diabetic retinopathy, and various types of cancer. We also reviewed the physiological roles of MACF1, including its involvement in adhesome formation, bone formation, neuronal aging, and tooth development. In addition, MACF1 plays other roles, functioning as a biomarker for the prediction of infections in patients with burns and as a marker for genome selection breeding. These studies reinforce the idea that MACF1 is a bona fide versatile, multifaceted giant protein. Identifying additional MACF1 functions would finally help with the treatment of diseases caused by MACF1 defects.

During gastrulation, the primitive streak (PS) forms and begins to differentiate into mesendodermal subtypes. This process involves an epithelial-mesenchymal transition (EMT), which is marked by cadherin switching, where E-Cadherin is downregulated, and N-Cadherin is upregulated. To understand the relationships between differentiation, EMT, and cadherin switching, we made measurements of these processes during differentiation of human pluripotent stem cells (hPSCs) to PS and subsequently to mesendoderm subtypes using established protocols, as well as variants in which signaling through key pathways including Activin, BMP, and Wnt were modulated. We found that perturbing signaling so that cells acquired identities ranging from anterior to posterior PS had little impact on the subsequent differentiation potential of cells but strongly impacted the degree of cadherin switching. The degree of E-Cadherin downregulation and N-Cadherin upregulation were uncorrelated and had different dependence on signaling. The exception to the broad potential of cells throughout the PS was the loss of definitive endoderm potential in cells with mid to posterior PS identities. Thus, cells induced to different PS coordinates had similar potential within the mesoderm but differed in cadherin switching. Consistently, E-Cadherin knockout did not alter cell fates outcomes during differentiation. Overall, cadherin switching and EMT are modulated independently of cell fate commitment in mesendodermal differentiation.

Blood vessels permeate all organs and execute myriad roles in health and disease. Here, we present a protocol to efficiently generate human artery and vein endothelial cells (ECs) from pluripotent stem cells within 3-4 days of differentiation. We delineate how to seed human pluripotent stem cells and sequentially differentiate them into primitive streak, lateral mesoderm, and either artery or vein ECs. We differentiate stem cells in defined, serum-free culture media in monolayers, without feeder cells or genetic manipulations. For complete details on the use and execution of this protocol, please refer to Ang et al. 1.

Hematopoietic stem cells (HSCs) generate blood and immune cells. Here, we present a protocol to differentiate human pluripotent stem cells (hPSCs) into hematopoietic progenitors that express the signature HSC transcription factors HLF, HOXA5, HOXA7, HOXA9, and HOXA10. hPSCs are dissociated, seeded, and then sequentially differentiated into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and hematopoietic progenitors through the sequential addition of defined, serum-free media. This 10-day protocol enables the manufacturing of blood and immune cells in monolayer cultures. For complete details on the use and execution of this protocol, please refer to Fowler and Zheng et al.1.

Induction of the neural ectoderm and the patterning of embryonic brain are the requisite organizing activity for head formation. Studies of loss-of-function mouse mutants that displayed a head truncation phenotype pointed to a key functional role of the anterior mesendoderm in anterior neural patterning. In this overview, we highlight the learning of the molecular attributes underpinning the formation of the anterior mesendoderm, the acquisition of ectoderm competence in the epiblast and the patterning of the embryonic brain during gastrulation and neurulation.

In chick embryos before primitive streak formation, the outermost extra-embryonic region, known as the area opaca (AO), was generally thought to act only by providing nutrients and mechanical support to the embryo. Immediately internal to the AO is a ring of epiblast called the marginal zone (MZ), separating the former from the inner area pellucida (AP) epiblast. The MZ does not contribute cells to any part of the embryo but is involved in determining the position of primitive streak formation from the adjacent AP epiblast. Recently, it was discovered that the AO can induce an MZ from AP epiblast. Here, we explore the nature of this inductive signal. We find that WNT8C is highly expressed in the AO, whereas canonical Wnt pathway targets are enriched in the MZ, along with strong nuclear β-catenin localization. Using isolation and recombination experiments combined with gain- and loss-of-function by exogenous chemical modulators of the pathway, we reveal that Wnt signalling is essential for induction and maintenance of the MZ, as well as sufficient to induce MZ properties in AP epiblast. We propose that canonical Wnt signalling is responsible for induction of the MZ by the area opaca.

Precise regulation of cell-cell communication is vital for cell survival and normal function during embryogenesis. The Wnt protein family, a highly conserved and extensively studied group, plays a crucial role in key cell-cell signaling events essential for development and regeneration. Congenital anomalies of the kidney and urinary tract (CAKUT) represent a leading cause of chronic kidney disease in children and young adults, and include a variety of birth abnormalities resulting from disrupted genitourinary tract development during embryonic development. The incidence and progression of CAKUT may be related to the Wnt signal transduction mechanism.

This review provides a comprehensive overview of the classical Wnt signaling pathway's role in CAKUT, explores related molecular mechanisms and provides new targets and intervention methods for the future treatment of the disease.

The Wnt signal is intricately engaged in a variety of differentiation processes throughout kidney development.

Epithelial-mesenchymal interactions (EMI) are fundamental mechanisms in regulating development and organogenesis. Here we summarized the signaling mechanisms involved in EMI in the major developmental events during craniofacial morphogenesis, including neural crest cell induction, facial primordial growth as well as fusion processes. Regional specificity/polarity are demonstrated in the expression of most signaling molecules that usually act in a mutually synergistic/antagonistic manner. The underlying mechanisms of pathogenesis due to disrupted EMI was also discussed in this review.

Breaking radial symmetry for anterior-posterior axis formation is one of the key developmental steps of vertebrate gastrulation and is established through a succession of transient domains defined by morphology or gene expression. Three such domains were interpreted recently in the rabbit to be part of a "three-anchor-point model" for axis formation. To answer the question as to whether the model is generally applicable to mammals, the dynamic expression patterns of four marker genes were analyzed in the pig, where gastrulating epiblast forms from half the inner cell mass: EOMES and PKDCC transcripts display decreasing expression intensities in the anterior hypoblast and-together with WNT3-increasing intensity in the anterior streak domain and the node; TBX6 expression changes from an initial central expression to exclusive expression in the posterior extremity of the primitive streak. The anterior streak domain has thus a molecular footprint similar to the one in the rabbit, the end node shares TBX6 between the species, while the anterior hypoblast-mirroring specific porcine epiblast derivation and fate-is marked by PKDCC instead of WNT3. The molecular similarities in transient domains point to conserved mechanisms for establishing the mammalian anterior-posterior axis and, possibly, breaking radial symmetry.

Embryonic stem cells (ESCs) hold great promise for regenerative medicine thanks to their ability to self-renew and differentiate into somatic cells and the germline. ESCs correspond to pluripotent epiblast - the tissue from which the following three germ layers originate during embryonic gastrulation: the ectoderm, mesoderm, and endoderm. Importantly, ESCs can be induced to differentiate toward various cell types by varying culture conditions, which can be exploited for in vitro modeling of developmental processes such as gastrulation. The classical model of gastrulation postulates that mesoderm and endoderm specification is made possible through the FGF-, BMP-, Wnt-, and Nodal-signaling gradients. Hence, it can be expected that one of these signals should direct ESC differentiation towards specific germ layers. However, ESC specification appears to be more complicated, and the same signal can be interpreted differently depending on the readout. In this research, using chemically defined culture conditions, homogeneous naïve ESCs as a starting cell population, and the Foxa2 gene-driven EGFP reporter tool, we established a robust model of definitive endoderm (DE) specification. This in vitro model features formative pluripotency as an intermediate state acquired by the epiblast in vivo shortly after implantation. Despite the initially homogeneous state of the cells in the model and high Activin concentration during endodermal specification, there remains a cell subpopulation that does not reach the endodermal state. This simple model developed by us can be used to study the origins of cellular heterogeneity during germ layer specification.

In the developing mammalian kidney, nephron formation is initiated by a subset of nephron progenitor cells (NPCs). Wnt input activates a β-catenin (Ctnnb1)-driven, transcriptional nephrogenic program and the mesenchymal to epithelial transition (MET) of NPCs. Using an in vitro mouse NPC culture model, we observed that activation of the Wnt pathway results in the aggregation of induced NPCs, which is an initiating step in the MET program. Genetic removal showed aggregation was dependent on β-catenin. Modulating extracellular Ca2+ levels showed cell-cell contacts were Ca2+ dependent, suggesting a role for cadherin (Cdh)-directed cell adhesion. Molecular analysis identified Cdh2, Cdh4 and Cdh11 in NPCs, and the β-catenin directed upregulation of Cdh3 and Cdh4 accompanying the MET of induced NPCs. Mutational analysis of β-catenin supported a role for a Lef/Tcf-β-catenin-mediated transcriptional response in the cell aggregation process. Genetic removal of all four cadherins, and independent removal of α-catenin or of β-catenin-α-catenin interactions, abolished aggregation, but not the inductive response to Wnt pathway activation. These findings, and data in an accompanying article highlight the role of β-catenin in linking transcriptional programs to the morphogenesis of NPCs in mammalian nephrogenesis.

How distinct mesodermal lineages - extraembryonic, lateral, intermediate, paraxial and axial - are specified from pluripotent epiblast during gastrulation is a longstanding open question. By investigating AXIN, a negative regulator of the WNT/β-catenin pathway, we have uncovered new roles for WNT signaling in the determination of mesodermal fates. We undertook complementary approaches to dissect the role of WNT signaling that augmented a detailed analysis of Axin1;Axin2 mutant mouse embryos, including single-cell and single-embryo transcriptomics, with in vitro pluripotent Epiblast-Like Cell differentiation assays. This strategy allowed us to reveal two layers of regulation. First, WNT initiates differentiation of primitive streak cells into mesoderm progenitors, and thereafter, WNT amplifies and cooperates with BMP/pSMAD1/5/9 or NODAL/pSMAD2/3 to propel differentiating mesoderm progenitors into either posterior streak derivatives or anterior streak derivatives, respectively. We propose that Axin1 and Axin2 prevent aberrant differentiation of pluripotent epiblast cells into mesoderm by spatially and temporally regulating WNT signaling levels.

Developmental hazard evaluation is an important part of assessing chemical risks during pregnancy. Toxicological outcomes from prenatal testing in pregnant animals result from complex chemical-biological interactions, and while New Approach Methods (NAMs) based on in vitro bioactivity profiles of human cells offer promising alternatives to animal testing, most of these assays lack cellular positional information, physical constraints, and regional organization of the intact embryo. Here, we engineered a fully computable model of the embryonic disc in the CompuCell3D.org modeling environment to simulate epithelial-mesenchymal transition (EMT) of epiblast cells and self-organization of mesodermal domains (chordamesoderm, paraxial, lateral plate, posterior/extraembryonic). Mesodermal fate is modeled by synthetic activity of the BMP4-NODAL-WNT signaling axis. Cell position in the epiblast determines timing with respect to EMT for 988 computational cells in the computer model. An autonomous homeobox (Hox) clock hidden in the epiblast is driven by WNT-FGF4-CDX signaling. Executing the model renders a quantitative cell-level computation of mesodermal fate and consequences of perturbation based on known biology. For example, synthetic perturbation of the control network rendered altered phenotypes (cybermorphs) mirroring some aspects of experimental mouse embryology, with electronic knockouts, under-activation (hypermorphs) or over-activation (hypermorphs) particularly affecting the size and specification of the posterior mesoderm. This foundational model is trained on embryology but capable of performing a wide variety of toxicological tasks conversing through anatomical simulation to integrate in vitro chemical bioactivity data with known embryology. It is amenable to quantitative simulation for probabilistic prediction of early developmental toxicity.

Animal body plans are established during embryonic development by the Hox genes. This patterning process relies on the differential expression of Hox genes along the head-to-tail axis. Hox spatial collinearity refers to the relationship between the organization of Hox genes in clusters and the differential Hox expression, whereby the relative order of the Hox genes within a cluster mirrors the spatial sequence of expression in the developing embryo. In vertebrates, the cluster organization is also associated with the timing of Hox activation, which harmonizes Hox expression with the progressive emergence of axial tissues. Thereby, in vertebrates, Hox temporal collinearity is intimately linked to Hox spatial collinearity. Understanding the mechanisms contributing to Hox temporal and spatial collinearity is thus key to the comprehension of vertebrate patterning. Here, we provide an overview of the main discoveries pertaining to the mechanisms of Hox spatial-temporal collinearity.

Wnts are secreted, lipid-modified proteins that bind to different receptors on the cell surface to activate canonical or non-canonical Wnt signaling pathways, which control various biological processes throughout embryonic development and adult life. Aberrant Wnt signaling pathway underlies a wide range of human disease pathogeneses. In this review, we provide an update of Wnt/β-catenin signaling components and mechanisms in bone formation, homeostasis, and diseases. The Wnt proteins, receptors, activators, inhibitors, and the crosstalk of Wnt signaling pathways with other signaling pathways are summarized and discussed. We mainly review Wnt signaling functions in bone formation, homeostasis, and related diseases, and summarize mouse models carrying genetic modifications of Wnt signaling components. Moreover, the therapeutic strategies for treating bone diseases by targeting Wnt signaling, including the extracellular molecules, cytosol components, and nuclear components of Wnt signaling are reviewed. In summary, this paper reviews our current understanding of the mechanisms by which Wnt signaling regulates bone formation, homeostasis, and the efforts targeting Wnt signaling for treating bone diseases. Finally, the paper evaluates the important questions in Wnt signaling to be further explored based on the progress of new biological analytical technologies.

DNA methyltransferases and Ten-Eleven Translocation (TET) proteins regulate the DNA methylation and demethylation cycles during mouse embryonic development. Although DNMT1 mainly plays a role in the maintenance of DNA methylation after DNA replication, it is also reported to possess de novo methyltransferase capacity. However, its physiological significance remains unclear. Here, we demonstrate that full-length DNMT1 (FL) and a mutant lacking the N-terminus necessary for its maintenance activity (602) confer the differentiation potential of mouse Dnmt1, Dnmt3a, and Dnmt3b (Dnmts-TKO) embryonic stem cells (ESCs). Both FL and 602 inhibit the spontaneous differentiation of Dnmts-TKO ESCs in the undifferentiated state. Dnmts-TKO ESCs showed loss of DNA methylation and de-repression of primitive endoderm-related genes, but these defects were partially restored in Dnmts-TKO + FL and Dnmts-TKO + 602 ESCs. Upon differentiation, Dnmts-TKO + FL ESCs show increased 5mC and 5hmC levels across chromosomes, including pericentromeric regions. In contrast, Dnmts-TKO + 602 ESCs didn't accumulate 5mC, and sister chromatids showed 5hmC asynchronously. Furthermore, in comparison with DNMT1_602, DNMT1_FL effectively promoted commitment to the epiblast-like cells and beyond, driving cell-autonomous mesendodermal and germline differentiation through embryoid body-based methods. With precise target selectivity achieved by its N-terminal region, DNMT1 may play a role in gene regulation leading to germline development.

Cardiomyocytes are the largest cell type that make up the heart and confer beating activity to the heart. The proper differentiation of cardiomyocytes relies on the efficient transmission and perception of differentiation cues from several signaling pathways that influence cardiomyocyte-specific gene expression programs. Signaling pathways also mediate intercellular communications to promote proper cardiomyocyte differentiation. We have reviewed the major signaling pathways involved in cardiomyocyte differentiation, including the BMP, Notch, sonic hedgehog, Hippo, and Wnt signaling pathways. Additionally, we highlight the differences between different cardiomyocyte cell lines and the use of these signaling pathways in the differentiation of cardiomyocytes from stem cells. Finally, we conclude by discussing open questions and current gaps in knowledge about the in vitro differentiation of cardiomyocytes and propose new avenues of research to fill those gaps.

Cell-fate decisions during mammalian gastrulation are poorly understood outside of rodent embryos. The embryonic disc of pig embryos mirrors humans, making them a useful proxy for studying gastrulation. Here we present a single-cell transcriptomic atlas of pig gastrulation, revealing cell-fate emergence dynamics, as well as conserved and divergent gene programs governing early porcine, primate, and murine development. We highlight heterochronicity in extraembryonic cell-types, despite the broad conservation of cell-type-specific transcriptional programs. We apply these findings in combination with functional investigations, to outline conserved spatial, molecular, and temporal events during definitive endoderm specification. We find early FOXA2 + /TBXT- embryonic disc cells directly form definitive endoderm, contrasting later-emerging FOXA2/TBXT+ node/notochord progenitors. Unlike mesoderm, none of these progenitors undergo epithelial-to-mesenchymal transition. Endoderm/Node fate hinges on balanced WNT and hypoblast-derived NODAL, which is extinguished upon endodermal differentiation. These findings emphasise the interplay between temporal and topological signalling in fate determination during gastrulation.

Pancreatic development is orchestrated by timely synthesis and degradation of stage-specific transcription factors (TFs). The transition from one stage to another stage is dependent on the precise expression of the developmentally relevant TFs. Persistent expression of particular TF would impede the exit from the progenitor stage to the matured cell type. Intracellular protein degradation-mediated protein turnover contributes to a major extent to the turnover of these TFs and thereby dictates the development of different tissues. Since even subtle changes in the crucial cellular pathways would dramatically impact pancreatic β-cell performance, it is generally acknowledged that the biological activity of these pathways is tightly regulated by protein synthesis and degradation process. Intracellular protein degradation is executed majorly by the ubiquitin proteasome system (UPS) and Lysosomal degradation pathway. As more than 90% of the TFs are targeted to proteasomal degradation, this review aims to examine the crucial role of UPS in normal pancreatic β-cell development and how dysfunction of these pathways manifests in metabolic syndromes such as diabetes. Such understanding would facilitate designing a faithful approach to obtain a therapeutic quality of β-cells from stem cells.

BMP signaling is essential for mammalian gastrulation, as it initiates a cascade of signals that control self-organized patterning. As development is highly dynamic, it is crucial to understand how time-dependent combinatorial signaling affects cellular differentiation. Here, we show that BMP signaling duration is a crucial control parameter that determines cell fates upon the exit from pluripotency through its interplay with the induced secondary signal WNT. BMP signaling directly converts cells from pluripotent to extraembryonic fates while simultaneously upregulating Wnt signaling, which promotes primitive streak and mesodermal specification. Using live-cell imaging of signaling and cell fate reporters together with a simple mathematical model, we show that this circuit produces a temporal morphogen effect where, once BMP signal duration is above a threshold for differentiation, intermediate and long pulses of BMP signaling produce specification of mesoderm and extraembryonic fates, respectively. Our results provide a systems-level picture of how these signaling pathways control the landscape of early human development.

The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.

Wnt/β-catenin signaling plays a major role in regulation of embryogenesis, organogenesis, and adult tissue homeostasis and regeneration. However, the roles played by Wnt/β-catenin and the spatiotemporal regulation of its activity throughout life, including during aging, are not fully understood. To address these issues, we introduced a Wnt/β-catenin signaling sensitive reporter into African turquoise killifish (Nothobranchius furzeri), a naturally ultra-short-lived fish that allows for the analysis of its whole life within a short period of time. Using this reporter killifish, we unraveled the previously unidentified dynamics of Wnt/β-catenin signaling during development and aging. Using the reporter strain, we detected Wnt/β-catenin activity in actively developing tissues as reported in previous reports, but also observed activation and attenuation of Wnt/β-catenin activity during embryonic reaggregation and diapause, respectively. During the aging process, the reporter was activated in the choroidal layer and liver, but its expression decreased in the kidneys. In addition, the reporter also revealed that aging disrupts the spatial regulation and intensity control of Wnt/β-catenin activity seen during fin regeneration, which interferes with precise regeneration. Thus, the employed reporter killifish is a highly useful model for investigating the dynamics of Wnt/β-catenin signaling during both the developmental and aging process.

Mammalian stem cell-based embryo models have emerged as innovative tools for investigating early embryogenesis in both mice and primates. They not only reduce the need for sacrificing mice but also overcome ethical limitations associated with human embryo research. Furthermore, they provide a platform to address scientific questions that are otherwise challenging to explore in vivo. The usefulness of a stem cell-based embryo model depends on its fidelity in replicating development, efficiency and reproducibility; all essential for addressing biological queries in a quantitative manner, enabling statistical analysis. Achieving such fidelity and efficiency requires robust systems that demand extensive optimization efforts. A profound understanding of pre- and post-implantation development, cellular plasticity, lineage specification, and existing models is imperative for making informed decisions in constructing these models. This review aims to highlight essential differences in embryo development and stem cell biology between mice and humans, assess how these variances influence the formation of partially and fully integrated stem cell models, and identify critical challenges in the field.

Cell therapies derived from induced pluripotent stem cells (iPSCs) offer a promising avenue in the field of regenerative medicine due to iPSCs' expandability, immune compatibility, and pluripotent potential. An increasing number of preclinical and clinical trials have been carried out, exploring the application of iPSC-based therapies for challenging diseases, such as muscular dystrophies. The unique syncytial nature of skeletal muscle allows stem/progenitor cells to integrate, forming new myonuclei and restoring the expression of genes affected by myopathies. This characteristic makes genome-editing techniques especially attractive in these therapies. With genetic modification and iPSC lineage specification methodologies, immune-compatible healthy iPSC-derived muscle cells can be manufactured to reverse the progression of muscle diseases or facilitate tissue regeneration. Despite this exciting advancement, much of the development of iPSC-based therapies for muscle diseases and tissue regeneration is limited to academic settings, with no successful clinical translation reported. The unknown differentiation process in vivo, potential tumorigenicity, and epigenetic abnormality of transplanted cells are preventing their clinical application. In this review, we give an overview on preclinical development of iPSC-derived myogenic cell transplantation therapies including processes related to iPSC-derived myogenic cells such as differentiation, scaling-up, delivery, and cGMP compliance. And we discuss the potential challenges of each step of clinical translation. Additionally, preclinical model systems for testing myogenic cells intended for clinical applications are described.

Wnt signaling is a critical determinant of cell lineage development. This study used Wnt dose-dependent induction programs to gain insights into molecular regulation of stem cell differentiation. We performed single-cell RNA sequencing of hiPSCs responding to a dose escalation protocol with Wnt agonist CHIR-99021 during the exit from pluripotency to identify cell types and genetic activity driven by Wnt stimulation. Results of activated gene sets and cell types were used to build a multiple regression model that predicts the efficiency of cardiomyocyte differentiation. Cross-referencing Wnt-associated gene expression profiles to the Connectivity Map database, we identified the small-molecule drug, tranilast. We found that tranilast synergistically activates Wnt signaling to promote cardiac lineage differentiation, which we validate by in vitro analysis of hiPSC differentiation and in vivo analysis of developing quail embryos. Our study provides an integrated workflow that links experimental datasets, prediction models, and small-molecule databases to identify drug-like compounds that control cell differentiation.

The Wnt pathway is essential for inducing the primitive streak, the precursor of the mesendoderm, as well as setting anterior-posterior coordinates. How Wnt coordinates these diverse activities remains incompletely understood. Here, we show that in Wnt-treated human pluripotent cells, endogenous Nodal signaling is a crucial switch between posteriorizing and primitive streak-including activities. While treatment with Wnt posteriorizes cells in standard culture, in micropatterned colonies, higher levels of endogenously induced Nodal signaling combine with exogenous Wnt to drive endoderm differentiation. Inhibition of Nodal signaling restores dose-dependent posteriorization by Wnt. In the absence of Nodal inhibition, micropatterned colonies undergo spontaneous, elaborate morphogenesis concomitant with endoderm differentiation even in the absence of added extracellular matrix proteins like Matrigel. Our study shows how Wnt and Nodal combinatorially coordinate germ layer differentiation with AP patterning and establishes a system to study a natural self-organizing morphogenetic event in in vitro culture.

Owing to their manifold roles in health and disease, there have been intense efforts to synthetically generate blood vessels in vitro from human pluripotent stem cells (hPSCs). However, there are multiple types of blood vessel, including arteries and veins, which are molecularly and functionally different. How can we specifically generate either arterial or venous endothelial cells (ECs) from hPSCs in vitro? Here, we summarize how arterial or venous ECs arise during embryonic development. VEGF and NOTCH arbitrate the bifurcation of arterial vs. venous ECs in vivo. While manipulating these two signaling pathways biases hPSC differentiation towards arterial and venous identities, efficiently generating these two subtypes of ECs has remained challenging until recently. Numerous questions remain to be fully addressed. What is the complete identity, timing and combination of extracellular signals that specify arterial vs. venous identities? How do these extracellular signals intersect with fluid flow to modulate arteriovenous fate? What is a unified definition for endothelial progenitors or angioblasts, and when do arterial vs. venous potentials segregate? How can we regulate hPSC-derived arterial and venous ECs in vitro, and generate organ-specific ECs? In turn, answers to these questions could avail the production of arterial and venous ECs from hPSCs, accelerating vascular research, tissue engineering, and regenerative medicine.

During embryonic development, the timing of events at the cellular level must be coordinated across multiple length scales to ensure the formation of a well-proportioned body plan. This is clear during somitogenesis, where progenitors must be allocated to the axis over time whilst maintaining a progenitor population for continued elaboration of the body plan. However, the relative importance of intrinsic and extrinsic signals in timing progenitor addition at the single-cell level is not yet understood. Heterochronic grafts from older to younger embryos have suggested a level of intrinsic timing whereby later staged cells contribute to more posterior portions of the axis. To determine the precise step at which cells are delayed, we performed single-cell transcriptomic analysis on heterochronic grafts of somite progenitors in the chicken embryo. This revealed a previously undescribed cell state within which heterochronic grafted cells are stalled. The delayed exit of older cells from this state correlates with expression of posterior Hox genes. Using grafting and explant culture, we find that both Hox gene expression and the migratory capabilities of progenitor populations are intrinsically regulated at the population level. However, by grafting varied sizes of tissue, we find that small heterochronic grafts disperse more readily and contribute to more anterior portions of the body axis while still maintaining Hox gene expression. This enhanced dispersion is not replicated in explant culture, suggesting that it is a consequence of interaction between host and donor tissue and thus extrinsic to the donor tissue. Therefore, we demonstrate that the timing of cell dispersion and resulting axis contribution is impacted by a combination of both intrinsic and extrinsic cues.

The freshwater cnidarian Hydra can regenerate from wounds, small tissue fragments and even from aggregated cells. This process requires the de novo development of a body axis and oral-aboral polarity, a fundamental developmental process that involves chemical patterning and mechanical shape changes. Gierer and Meinhardt recognized that Hydra's simple body plan and amenability to in vivo experiments make it an experimentally and mathematically tractable model to study developmental patterning and symmetry breaking. They developed a reaction-diffusion model, involving a short-range activator and a long-range inhibitor, which successfully explained patterning in the adult animal. In 2011, HyWnt3 was identified as a candidate for the activator. However, despite the continued efforts of both physicists and biologists, the predicted inhibitor remains elusive. Furthermore, the Gierer-Meinhardt model cannot explain de novo axis formation in cellular aggregates that lack inherited tissue polarity. The aim of this review is to synthesize the current knowledge on Hydra symmetry breaking and patterning. We summarize the history of patterning studies and insights from recent biomechanical and molecular studies, and highlight the need for continued validation of theoretical assumptions and collaboration across disciplinary boundaries. We conclude by proposing new experiments to test current mechano-chemical coupling models and suggest ideas for expanding the Gierer-Meinhardt model to explain de novo patterning, as observed in Hydra aggregates. The availability of a fully sequenced genome, transgenic fluorescent reporter strains, and modern imaging techniques, that enable unprecedented observation of cellular events in vivo, promise to allow the community to crack Hydra's secret to patterning.

Gastrulation represents a pivotal phase of development and aberrations during this period can have major consequences, from minor anatomical deviations to severe congenital defects. Animal models are used to study gastrulation, however, there is considerable morphological and molecular diversity of gastrula across mammalian species. Here, we provide an overview of the latest research on interspecies developmental control across mammals. This includes single-cell atlases of several mammalian gastrula which have enabled comparisons of the temporal and molecular dynamics of differentiation. These studies highlight conserved cell differentiation regulators and both absolute and relative differences in differentiation dynamics between species. Recent advances in in vitro culture techniques have facilitated the derivation, maintenance and differentiation of cell lines from a range of species and the creation of multi-species models of gastrulation. Gastruloids are three-dimensional aggregates capable of self-organising and recapitulating aspects of gastrulation. Such models enable species comparisons outside the confines of the embryo. We highlight recent in vitro evidence that differentiation processes such as somitogenesis and neuronal maturation scale with known in vivo differences in developmental tempo across species. This scaling is likely due to intrinsic differences in cell biochemistry. We also highlight several studies which provide examples of cell differentiation dynamics being influenced by extrinsic factors, including culture conditions, chimeric co-culture, and xenotransplantation. These collective studies underscore the complexity of gastrulation across species, highlighting the necessity of additional datasets and studies to decipher the intricate balance between intrinsic cellular programs and extrinsic signals in shaping embryogenesis.

The development of the vertebrate spinal cord involves the formation of the neural tube and the generation of multiple distinct cell types. The process starts during gastrulation, combining axial elongation with specification of neural cells and the formation of the neuroepithelium. Tissue movements produce the neural tube which is then exposed to signals that provide patterning information to neural progenitors. The intracellular response to these signals, via a gene regulatory network, governs the spatial and temporal differentiation of progenitors into specific cell types, facilitating the assembly of functional neuronal circuits. The interplay between the gene regulatory network, cell movement, and tissue mechanics generates the conserved neural tube pattern observed across species. In this review we offer an overview of the molecular and cellular processes governing the formation and patterning of the neural tube, highlighting how the remarkable complexity and precision of vertebrate nervous system arises. We argue that a multidisciplinary and multiscale understanding of the neural tube development, paired with the study of species-specific strategies, will be crucial to tackle the open questions.

Although vertebrates display a large variety of forms and sizes, the mechanisms controlling the layout of the basic body plan are substantially conserved throughout the clade. Following gastrulation, head, trunk, and tail are sequentially generated through the continuous addition of tissue at the caudal embryonic end. Development of each of these major embryonic regions is regulated by a distinct genetic network. The transitions from head-to-trunk and from trunk-to-tail development thus involve major changes in regulatory mechanisms, requiring proper coordination to guarantee smooth progression of embryonic development. In this review, we will discuss the key cellular and embryological events associated with those transitions giving particular attention to their regulation, aiming to provide a cohesive outlook of this important component of vertebrate development.

Primordial germ cell (PGC) specification is the first step in the development of the germline. Recent work has elucidated human-mouse differences in PGC differentiation and identified cell states with enhanced competency for PGC-like cell (PGCLC) differentiation in vitro in both species. However, it remains a subject of debate how different PGC competent states in vitro relate to each other, to embryonic development, and to the origin of PGCs in vivo. Here we review recent literature on human PGCLC differentiation in the context of mouse and non-human primate models. In contrast to what was previously thought, recent work suggests human pluripotent stem cells (hPSCs) are highly germline competent. We argue that paradoxical observations regarding the origin and signaling requirements of hPGCLCs may be due to local cell interactions. These confound assays of competence by generating endogenous signaling gradients and spatially modulating the ability to receive exogenous inductive signals. Furthermore, combinatorial signaling suggests that there is no unique germline competent state: rather than a one-dimensional spectrum of developmental progression, competence should be considered in a higher dimensional landscape of cell states.

Obesity is the leading risk factor for the development of type 2 diabetes and cardiovascular diseases. Childhood obesity represents an alarming health challenge because children with obesity are prone to remain with obesity throughout their life and have an increased morbidity and mortality risk. The ability of adipose tissue to store lipids and expand in size during excessive calorie intake is its most remarkable characteristic. Cellular and lipid turnovers determine adipose tissue size and are closely related with metabolic status. The mechanisms through which adipose tissue expands and how this affects systemic metabolic homeostasis are still poorly characterized. Furthermore, the mechanism through which increased adiposity extends from childhood to adulthood and its implications in metabolic health are in most part, still unknown. More studies on adipose tissue development in healthy and children with obesity are urgently needed. In the present review, we summarize the dynamics of white adipose tissue, from developmental origins to the mechanisms that allows it to grow and expand throughout lifetime and during obesity in children and in different mouse models used to address this largely unknown field. Specially, highlighting the role that excessive adiposity during the early life has on future's adipose tissue dynamics and individual's health.

Adrenal insufficiency is a life-threatening condition resulting from the inability to produce adrenal hormones in a dose- and time-dependent manner. Establishing a cell-based therapy would provide a physiologically responsive approach for the treatment of this condition. We report the generation of large numbers of human-induced steroidogenic cells (hiSCs) from human pluripotent stem cells (hPSCs). Directed differentiation of hPSCs into hiSCs recapitulates the initial stages of human adrenal development. Following expression of steroidogenic factor 1, activation of protein kinase A signaling drives a steroidogenic gene expression profile most comparable to human fetal adrenal cells, and leads to dynamic secretion of steroid hormones, in vitro. Moreover, expression of the adrenocorticotrophic hormone (ACTH) receptor/co-receptor (MC2R/MRAP) results in dose-dependent ACTH responsiveness. This protocol recapitulates adrenal insufficiency resulting from loss-of-function mutations in AAAS, which cause the enigmatic triple A syndrome. Our differentiation protocol generates sufficient numbers of hiSCs for cell-based therapy and offers a platform to study disorders causing adrenal insufficiency.

The origin of the pentaradial body plan of echinoderms from a bilateral ancestor is one of the most enduring zoological puzzles1,2. Because echinoderms are defined by morphological novelty, even the most basic axial comparisons with their bilaterian relatives are problematic. To revisit this classical question, we used conserved anteroposterior axial molecular markers to determine whether the highly derived adult body plan of echinoderms masks underlying patterning similarities with other deuterostomes. We investigated the expression of a suite of conserved transcription factors with well-established roles in the establishment of anteroposterior polarity in deuterostomes3-5 and other bilaterians6-8 using RNA tomography and in situ hybridization in the sea star Patiria miniata. The relative spatial expression of these markers in P. miniata ambulacral ectoderm shows similarity with other deuterostomes, with the midline of each ray representing the most anterior territory and the most lateral parts exhibiting a more posterior identity. Strikingly, there is no ectodermal territory in the sea star that expresses the characteristic bilaterian trunk genetic patterning programme. This finding suggests that from the perspective of ectoderm patterning, echinoderms are mostly head-like animals and provides a developmental rationale for the re-evaluation of the events that led to the evolution of the derived adult body plan of echinoderms.

Stem-cell-based embryo models have allowed greater insight into peri-implantation mammalian developmental events that are otherwise difficult to manipulate due to the inaccessibility of the early embryo. The rapid development of this field has resulted in the precise roles of frequently used supplements such as N2, B27 and Chiron in driving stem cell lineage commitment not being clearly defined. Here, we investigate the effects of these supplements on embryoid bodies to better understand their roles in stem cell differentiation. We show that Wnt signalling has a general posteriorising effect on stem cell aggregates and directs differentiation towards the mesoderm, as confirmed through the upregulation of posterior and mesodermal markers. N2 and B27 can mitigate these effects and upregulate the expression of anterior markers. To control the Wnt gradient and the subsequent anterior versus posterior fate, we make use of a BMP4 signalling centre and show that aggregates in these conditions express cephalic markers. These findings indicate that there is an intricate balance between various culture supplements and their ability to guide differentiation in stem cell embryo models.

Naive human pluripotent stem cells have the remarkable ability to self-organize into blastocyst-like structures ("blastoids") that model lineage segregation in the pre-implantation embryo. However, the extent to which blastoids can recapitulate the defining features of human post-implantation development remains unexplored. Here, we report that blastoids cultured on thick three-dimensional (3D) extracellular matrices capture hallmarks of early post-implantation development, including epiblast lumenogenesis, rapid expansion and diversification of trophoblast lineages, and robust invasion of extravillous trophoblast cells by day 14. Extended blastoid culture results in the localized activation of primitive streak marker TBXT and the emergence of embryonic germ layers by day 21. We also show that the modulation of WNT signaling alters the balance between epiblast and trophoblast fates in post-implantation blastoids. This work demonstrates that 3D-cultured blastoids offer a continuous and integrated in vitro model system of human embryonic and extraembryonic development from pre-implantation to early gastrulation stages.

During early mammalian embryonic development, fertilized one-cell embryos develop into pre-implantation blastocysts and subsequently establish three germ layers through gastrulation during post-implantation development. In recent years, stem cells have emerged as a powerful tool to study embryogenesis and gastrulation without the need for eggs, allowing for the generation of embryo-like structures known as synthetic embryos or embryoids. These in vitro models closely resemble early embryos in terms of morphology and gene expression and provide a faithful recapitulation of early pre- and post-implantation embryonic development. Synthetic embryos can be generated through a combinatorial culture of three blastocyst-derived stem cell types, such as embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm cells, or totipotent-like stem cells alone. This review provides an overview of the progress and various approaches in studying in vitro embryogenesis and gastrulation in mice and humans using stem cells. Furthermore, recent findings and breakthroughs in synthetic embryos and gastruloids are outlined. Despite ethical considerations, synthetic embryo models hold promise for understanding mammalian (including humans) embryonic development and have potential implications for regenerative medicine and developmental research.

Male germ cells undergo a complex sequence of developmental events throughout fetal and postnatal life that culminate in the formation of haploid gametes: the spermatozoa. Errors in these processes result in infertility and congenital abnormalities in offspring. Male germ cell development starts when pluripotent cells undergo specification to sexually uncommitted primordial germ cells, which act as precursors of both oocytes and spermatozoa. Male-specific development subsequently occurs in the fetal testes, resulting in the formation of spermatogonial stem cells: the foundational stem cells responsible for lifelong generation of spermatozoa. Although deciphering such developmental processes is challenging in humans, recent studies using various models and single-cell sequencing approaches have shed new insight into human male germ cell development. Here, we provide an overview of cellular, signaling and epigenetic cascades of events accompanying male gametogenesis, highlighting conserved features and the differences between humans and other model organisms.

In the developing mammalian kidney, nephron formation is initiated by a subset of nephron progenitor cells (NPCs). Wnt input activates a β-catenin ( Ctnnb1 )-driven, transcriptional nephrogenic program. In conjunction, induced mesenchymal NPCs transition through a pre-tubular aggregate to an epithelial renal vesicle, the precursor for each nephron. How this critical mesenchymal-to-epithelial transition (MET) is regulated is unclear. In an in vitro mouse NPC culture model, activation of the Wnt pathway results in the aggregation of induced NPCs into closely-packed, cell clusters. Genetic removal of β-catenin resulted in a failure of both Wnt pathway-directed transcriptional activation and the formation of aggregated cell clusters. Modulating extracellular Ca 2+ levels showed cell-cell contacts were Ca 2+ -dependent, suggesting a role for cadherin (Cdh)-directed cell adhesion. Molecular analysis identified Cdh2 , Cdh4 and Cdh11 in uninduced NPCs and the up-regulation of Cdh3 and Cdh4 accompanying the Wnt pathway-induced MET. Genetic removal of all four cadherins, and independent removal of α-catenin, which couples Cdh-β-catenin membrane complexes to the actin cytoskeleton, abolished cell aggregation in response to Wnt pathway activation. However, the β-catenin driven inductive transcriptional program was unaltered. Together with the accompanying paper (Bugacov et al ., submitted), these data demonstrate that distinct cellular activities of β-catenin - transcriptional regulation and cell adhesion - combine in the mammalian kidney programs generating differentiated epithelial nephron precursors from mesenchymal nephron progenitors.

Our study highlights the role of Wnt-β-catenin pathway regulation of cadherin-mediated cell adhesion in the mesenchymal to epithelial transition of induced nephron progenitor cells.

Development of vertebrate embryos is characterized by early formation of the anterior tissues followed by the sequential extension of the axis at their posterior end to build the trunk and tail structures, first by the activity of the primitive streak and then of the tail bud. Embryological, molecular and genetic data indicate that head and trunk development are significantly different, suggesting that the transition into the trunk formation stage involves major changes in regulatory gene networks.

We explored those regulatory changes by generating differential interaction networks and chromatin accessibility profiles from the posterior epiblast region of mouse embryos at embryonic day (E)7.5 and E8.5. We observed changes in various cell processes, including several signaling pathways, ubiquitination machinery, ion dynamics and metabolic processes involving lipids that could contribute to the functional switch in the progenitor region of the embryo. We further explored the functional impact of changes observed in Wnt signaling associated processes, revealing a switch in the functional relevance of Wnt molecule palmitoleoylation, essential during gastrulation but becoming differentially required for the control of axial extension and progenitor differentiation processes during trunk formation. We also found substantial changes in chromatin accessibility at the two developmental stages, mostly mapping to intergenic regions and presenting differential footprinting profiles to several key transcription factors, indicating a significant switch in the regulatory elements controlling head or trunk development. Those chromatin changes are largely independent of retinoic acid, despite the key role of this factor in the transition to trunk development. We also tested the functional relevance of potential enhancers identified in the accessibility assays that reproduced the expression profiles of genes involved in the transition. Deletion of these regions by genome editing had limited effect on the expression of those genes, suggesting the existence of redundant enhancers that guarantee robust expression patterns.

This work provides a global view of the regulatory changes controlling the switch into the axial extension phase of vertebrate embryonic development. It also revealed mechanisms by which the cellular context influences the activity of regulatory factors, channeling them to implement one of several possible biological outputs.